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As a phosphorus-containing molecule, nicotinamide adenine dinucleotide is visible by phosphorus magnetic resonance spectroscopy (

Importance of the redox status of nicotinamide adenine dinucleotide (NAD), including its oxidized (NAD

Co-enzyme nicotinamide adenine dinucleotide NAD(H) regulates hundreds of biochemical reactions within the cell. We previously reported that NAD(H) redox status may have prognostic value for predicting breast cancer metastasis. However, the mechanisms of NAD(H) involvement in metastasis remain elusive. Given the important roles of TGFβ signalling in metastatic processes, such as promoting the epithelial-to-mesenchymal transition, we aimed to investigate the involvement of the mitochondrial NAD(H) redox status in TGFβ receptor signalling. Here we present the initial evidence that NAD(H) redox status is responsive to TGFβ receptor signalling in triple-negative breast cancer cells in culture. The mitochondrial NAD(H) redox status was determined by the optical redox imaging (ORI) technique. Cultured HCC1806 (less aggressive) and MDA-MB-231 (more aggressive) cells were subjected to ORI after treatment with exogenous TGFβ1 or LY2109761, which stimulates or inhibits TGFβ receptor signalling, respectively. Cell migration was determined with the transwell migration assay. Global averaging quantification of the ORI images showed that 1) TGFβ1 stimulation resulted in differential responses between HCC1806 and MDA-MB-231 lines, with HCC1806 cells having a significant change in the mitochondrial redox status, corresponding to a larger increase in cell migration; 2) HCC1806 cells acutely treated with LY2109761 yielded immediate increases in ORI signals. These preliminary data are the first evidence that suggests the existence of a cell line-dependent shift of the mitochondrial NAD(H) redox status in the TGFβ receptor signalling induced migratory process of breast cancer cells. Further research should be conducted to confirm these results as improved understanding of the underlying mechanisms of metastatic process may contribute to the identification of prognostic biomarkers and therapeutic targets.

Rapamycin delays multiple age-related conditions and extends lifespan in organisms ranging from yeast to mice. However, the mechanisms by which rapamycin influences longevity are incompletely understood. The objective of this study was to investigate the effect of rapamycin on NAD+/NADH redox balance. We report that the NAD+/NADH ratio of C2C12 myoblasts or differentiated myotubes significantly decreases over time in culture, and that rapamycin prevents this effect. Despite lowering the NADH available to support ATP generation, rapamycin increases ATP availability, consistent with lowering energetic demand. Although rapamycin did not change the NAD+/NADH ratio or steady-state ATP concentration in the livers, kidneys, or muscles of young mice, optical redox imaging revealed that rapamycin caused a substantial decline in the NADH content and an increase in the optical redox ratio (a surrogate of NAD+/NADH redox ratio) in muscles from aged mice. Collectively, these data suggest that rapamycin favors a more oxidized NAD+/NADH ratio in aged muscle, which may influence metabolism and the activity of NAD+-dependent enzymes. This study provides new insight into the mechanisms by which rapamycin might influence the aging process to improve health and longevity among the aging population.

Stratification of malignancy is valuable for cancer treatment. Both optical redox imaging (ORI) indices and nuclear-to-cytoplasmic volume/area ratio (N:C ratio) have been investigated to differentiate between cancers with varying aggressiveness, but these two methods have not been directly compared. The redox status in the cell nucleus has not been studied by ORI, and it remains unknown whether nuclear ORI indices add new biological information.We sought to compare the capacity of whole-cell and subcellular ORI indices and N:C ratio to differentiate between breast cancer subtypes with varying aggressiveness and between mitotic and nonmitotic cells.ORI indices for whole cell, cytoplasm, and nucleus as well as the N:C area ratio were generated for two triple-negative (more aggressive) and two receptor-positive (less aggressive) breast cancer cell lines by fluorescence microscopy.We found positive correlations between nuclear and cytoplasmic ORI indices within individual cells. On average, a nuclear redox status was found to be more oxidized than cytoplasm in triple-negative cells but not in receptor-positive cells. Whole-cell and subcellular ORI indices distinguished between the receptor statuses better than the N:C ratio. However, N:C ratio was a better differentiator between nonmitotic and mitotic triple-negative cells.Subcellular ORI analysis differentiates breast cancer subtypes with varying aggressiveness better than N:C area ratio.

PURPOSE: Optical redox imaging (ORI) technique images cellular autofluorescence of nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp containing FAD, i.e., flavin adenine dinucleotide). ORI has found wide applications in the study of cellular energetics and metabolism and may potentially assist in disease diagnosis and prognosis. Fixed tissues have been reported to exhibit autofluorescence with similar spectral characteristics to those of NADH and Fp. However, few studies report on quantitative ORI of formalin-fixed paraffin-embedded (FFPE) unstained tissue slides for disease biomarkers. We investigate whether ORI of FFPE unstained skeletal muscle slides may provide relevant quantitative biological information. PROCEDURES: Living mouse muscle fibers and frozen and FFPE mouse muscle slides were subjected to ORI. Living mouse muscle fibers were imaged ex vivo before and after paraformaldehyde fixation. FFPE muscle slides of three mouse groups (young, mid-age, and muscle-specific overexpression of nicotinamide phosphoribosyltransferase (Nampt) transgenic mid-age) were imaged and compared to detect age-related redox differences. RESULTS: We observed that living muscle fiber and frozen and FFPE slides all had strong autofluorescence signals in the NADH and Fp channels. Paraformaldehyde fixation resulted in a significant increase in the redox ratio Fp/(NADH + Fp) of muscle fibers. Quantitative image analysis on FFPE unstained slides showed that mid-age gastrocnemius muscles had stronger NADH and Fp signals than young muscles. Gastrocnemius muscles from mid-age Nampt mice had lower NADH compared to age-matched controls, but had higher Fp than young controls. Soleus muscles had the same trend of change and appeared to be more oxidative than gastrocnemius muscles. Differential NADH and Fp signals were found between gastrocnemius and soleus muscles within both mid-aged control and Nampt groups. CONCLUSION: Aging effect on redox status quantified by ORI of FFPE unstained muscle slides was reported for the first time. Quantitative information from ORI of FFPE unstained slides may be useful for biomedical applications.

We evaluated the utility of optical redox imaging (ORI) to identify the therapeutic response of triple-negative breast cancers (TNBC) under various drug treatments. Cultured HCC1806 and MDA-MB-231 cells treated with FK866 (nicotinamide phosphoribosyltransferase (Nampt) inhibitor), FX11 (lactate dehydrogenase A inhibitor), paclitaxel, and their combinations were subjected to ORI, followed by imaging fluorescently labeled reactive oxygen species (ROS). Cell growth inhibition was measured by a cell viability assay. We found that both cell lines experienced significant NADH decrease and redox ratio (Fp/(NADH+Fp)) increase due to FK866 treatment, however, HCC1806 was much more responsive than MDA-MB-231. We further studied HCC1806 with the main findings: (i) nicotinamide riboside (NR) partially restored NADH in FK866-treated cells, (ii) FX11 induced an over 3-fold NADH increase in FK866 or FK866+NR pretreated cells, (iii) FK866 combined with paclitaxel caused synergistic increases in both Fp and the redox ratio, (iv) FK866 sensitized cells to paclitaxel treatments, which agrees with the redox changes detected by ORI, (v) Fp and the redox ratio positively correlated with cell growth inhibition, and (vi) Fp and NADH positively correlated with ROS level. Our study supports the utility of ORI for detecting the treatment responses of TNBC to Nampt inhibition and the sensitization effects on standard chemotherapeutics.

Triple-negative breast cancer (TNBC) is a highly diverse group of cancers with limited treatment options, responsible for about 15% of all breast cancers. TNBC cells differ from each other in many ways such as gene expression, metabolic activity, tumorigenicity, and invasiveness. Recently, many research and clinical efforts have focused on metabolically targeted therapy for TNBC. Metabolic characterization of TNBC cell lines can facilitate the assessment of therapeutic effects and assist in metabolic drug development. Herein, we used optical redox imaging (ORI) techniques to characterize TNBC subtypes metabolically. We found that various TNBC cell lines had differing redox statuses (levels of reduced nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD), and the redox ratio (FAD/(NADH+FAD)). We then metabolically perturbed the cells with mitochondrial inhibitors and an uncoupler and performed ORI accordingly. As expected, we observed that these TNBC cell lines had similar response patterns to the metabolic perturbations. However, they exhibited differing redox plasticity. These results suggest that subtypes of TNBC cells are different metabolically and that ORI can serve as a sensitive technique for the metabolic profiling of TNBC cells.

Dimethyl fumarate (DMF), an antioxidant/anti-inflammatory drug approved for the treatment of multiple sclerosis, induces antioxidant enzymes, in part through transcriptional upregulation. We hypothesized that DMF administration to simian immunodeficiency virus (SIV)-infected rhesus macaques would induce antioxidant enzyme expression and reduce oxidative injury and inflammation throughout the brain. Nine SIV-infected, CD8+-T-lymphocyte-depleted rhesus macaques were studied. Five received oral DMF prior to the SIV infection and through to the necropsy day. Protein expression was analyzed in 11 brain regions, as well as the thymus, liver, and spleen, using Western blot and immunohistochemistry for antioxidant, inflammatory, and neuronal proteins. Additionally, oxidative stress was determined in brain sections using immunohistochemistry (8-OHdG, 3NT) and optical redox imaging of oxidized flavoproteins containing flavin adenine dinucleotide (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The DMF treatment was associated with no changes in virus replication, higher expressions of the antioxidant enzymes NQO1, GPX1, and HO-1 in the brain and PRDX1 and HO-2 in the spleen, lower levels of 8-OHdG and 3NT, a lower optical redox ratio. The DMF treatment was also associated with increased expressions of cell-adhesion molecules (VCAM-1, ICAM-1) and no changes in HLA-DR, CD68, GFAP, NFL, or synaptic proteins. The concordantly increased brain antioxidant enzyme expressions and reduced oxidative stress in DMF-treated SIV-infected macaques suggest that DMF could limit oxidative stress throughout the brain through effective induction of the endogenous antioxidant response. We propose that DMF could potentially induce neuroprotective brain responses in persons living with HIV.

The Warburg effect, representing enhanced glycolysis and lactate production in adequately oxygenated cancer cells, has been widely regarded to cause increased extracellular acidification. Converting pyruvate to lactate by lactate dehydrogenase A (LDHA) is the last step of glycolysis. Here, we report an interesting counterintuitive observation that inhibition of LDHA resulted in enhanced glycolysis in MDA-MB-231 breast cancer cells. The cells were treated with FX11 (7-benzyl-2,3-dihydroxy-6-methyl-4-propylnaphthalene-1-carboxylic acid), a specific LDHA inhibitor. Seahorse assay reported dose-dependent increases in both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Independent biochemical measurements also confirmed the increase of lactate production under FX11 treatment. The reasons and mechanism of these observations of elevated ECAR and lactate production in the MDA-MB-231 breast cancer cells under FX11 treatment remain to be investigated.

Predicting tumor metastatic potential remains a challenge in cancer research and in clinical diagnosis. Cancer invasion to neighboring tissues is a significant event in cancer progression to metastasis. Optical redox imaging (ORI) is based on detecting the endogenous fluorescence signals of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD). Previously, we found that ORI can discriminate between cancer and normal tissue specimens from clinical breast cancer patients and can differentiate the relative invasiveness of melanoma and breast tumors. In this study, we aimed to identify ORI biomarkers to differentiate the invasiveness of four triple-negative breast cancer cell lines (TNBC). Using a fluorescence microscope, we acquired NADH and FAD fluorescent signals from cultured MDA-MB-231, MDA-MB-436, HCC1806, and MDA-MB-468 cells. We found that (1) the redox ratio, FAD/(NADH+FAD), differentiated the four TNBC lines; (2) there was a significant difference of invasive potential between MDA-MB-231 and the other three TNBC lines measured by the transwell invasion assay; and (3) there was a positive logarithmic correlation between the redox ratio and the invasive potential, where the most invasive MDA-MB-231 cells had the highest redox ratio and the least invasive MDA-MB-468 cells had the lowest redox ratio. These results suggest that the redox ratio can potentially be used as a biomarker for TNBC invasiveness and prognosis.

Triple-negative breast cancer (TNBC) is a highly diverse group of cancers with limited treatment options, responsible for about 15% of all breast cancers. TNBC cells differ from each other in many ways such as gene expression, metabolic activity, tumorigenicity, and invasiveness. Recently, many research and clinical efforts have focused on metabolically targeted therapy for TNBC. Metabolic characterization of TNBC cell lines can facilitate the assessment of therapeutic effects and assist in metabolic drug development. Herein, we used optical redox imaging (ORI) techniques to characterize TNBC subtypes metabolically. We found that various TNBC cell lines had differing redox statuses (levels of reduced nicotinamide adenine dinucleotide (NADH), oxidized flavin adenine dinucleotide (FAD), and the redox ratio (FAD/(NADH+FAD)). We then metabolically perturbed the cells with mitochondrial inhibitors and an uncoupler and performed ORI accordingly. As expected, we observed that these TNBC cell lines had similar response patterns to the metabolic perturbations. However, they exhibited differing redox plasticity. These results suggest that subtypes of TNBC cells are different metabolically and that ORI can serve as a sensitive technique for the metabolic profiling of TNBC cells.

Co-enzyme nicotinamide adenine dinucleotide (NAD(H)) redox plays a key role in macrophage function. Surfactant protein (SP-) A modulates the functions of alveolar macrophages (AM) and ozone (O3) exposure in the presence or absence of SP-A and reduces mouse survival in a sex-dependent manner. It is unclear whether and how NAD(H) redox status plays a role in the innate immune response in a sex-dependent manner. We investigated the NAD(H) redox status of AM from SP-A2 and SP-A knockout (KO) mice in response to O3 or filtered air (control) exposure using optical redox imaging technique. We found: (i) In SP-A2 mice, the redox alteration of AM in response to O3 showed sex-dependence with AM from males being significantly more oxidized and having a higher level of mitochondrial reactive oxygen species than females; (ii) AM from KO mice were more oxidized after O3 exposure and showed no sex differences; (iii) AM from female KO mice were more oxidized than female SP-A2 mice; and (iv) Two distinct subpopulations characterized by size and redox status were observed in a mouse AM sample. In conclusions, the NAD(H) redox balance in AM responds to O3 in a sex-dependent manner and the innate immune molecule, SP-A2, contributes to this observed sex-specific redox response.

PURPOSE: Optical redox imaging (ORI), based on collecting the endogenous fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) containing a redox cofactor flavin adenine dinucleotide (FAD), provides sensitive indicators of cellular metabolism and redox status. ORI indices (such as NADH, FAD, and their ratio) have been under investigation as potential progression/prognosis biomarkers for cancer. Higher FAD redox ratio, i.e., (FAD/(FAD+NADH)) has been associated with higher invasive/metastatic potential in tumor xenografts and cultured cells. This study is to examine whether ORI indices can respond to the modulation of oncogene DEK activities that change cancer cell invasive/metastatic potential. PROCEDURES: Using lentiviral shRNA, DEK gene expression was efficiently knocked down in MDA-MB-231 breast cancer cells (DEKsh). These DEKsh cells, along with scrambled shRNA transduced control cells (NTsh), were imaged with a fluorescence microscope. In vitro invasive potential of the DEKsh cells and NTsh cells were also measured in parallel using the transwell assay. RESULTS: FAD and FAD redox ratio in polyclonal cells with DEKsh were significantly lower than that in NTsh control cells. Consistently, the DEKsh cells demonstrated decreased invasive potential than their non-knockdown counterparts NTsh cells. CONCLUSIONS: This study provides direct evidence that oncogene activities could mediate ORI-detected cellular redox state.

Rapamycin delays multiple age-related conditions and extends lifespan in organisms ranging from yeast to mice. However, the mechanisms by which rapamycin influences longevity are incompletely understood. The objective of this study was to investigate the effect of rapamycin on NAD+/NADH redox balance. We report that the NAD+/NADH ratio of C2C12 myoblasts or differentiated myotubes significantly decreases over time in culture, and that rapamycin prevents this effect. Despite lowering the NADH available to support ATP generation, rapamycin increases ATP availability, consistent with lowering energetic demand. Although rapamycin did not change the NAD+/NADH ratio or steady-state ATP concentration in the livers, kidneys, or muscles of young mice, optical redox imaging revealed that rapamycin caused a substantial decline in the NADH content and an increase in the optical redox ratio (a surrogate of NAD+/NADH redox ratio) in muscles from aged mice. Collectively, these data suggest that rapamycin favors a more oxidized NAD+/NADH ratio in aged muscle, which may influence metabolism and the activity of NAD+-dependent enzymes. This study provides new insight into the mechanisms by which rapamycin might influence the aging process to improve health and longevity among the aging population.

Shifted NAD(H) redox status and enhanced reactive oxygen species (ROS) scavenging systems have been observed in cancers. However, how such redox shift is related to the ROS level in cancer cells is less clear. Based on collecting the intrinsic fluorescence of oxidized flavoproteins (Fp containing flavin adenine dinucleotide) and reduced nicotinamide adenine dinucleotide (NADH), optical redox imaging (ORI) provides a quantitative measure of the mitochondrial redox state by the optical redox ratio, Fp/(NADH+Fp), a surrogate marker of the NAD(+)-coupled redox state NAD(+)/NADH. Our study aims to explore the relationship between NAD(H) redox status and ROS by imaging NADH, Fp, and ROS levels using cultured breast cancer cell models. By manipulating either ROS levels via application of exogenous H(2)O(2) or redox status via metabolic perturbation compounds, we found that: (1) oxidation of NAD(H) redox status correlates with ROS levels at lower H(2)O(2) concentrations (up to ~700 μM), but not necessarily at higher concentrations; (2) an elevated ROS level diminishes NADH and reduces redox ratio plasticity; (3) either more oxidized or more reduced status can correlate to an increased ROS level; and (4) sometimes, a more oxidized status can correlate to a decreased ROS level depending on cell lines. These observations indicated that cellular NAD(H) redox state and ROS are intricately related but can also change separately. This study can benefit cancer research as both NAD(H) redox status and ROS have been implicated in cancer transformation and progression.

The value of optical redox imaging (ORI) of cells/tissues based on the intrinsic fluorescences of NADH (nicotinamide adenine dinucleotide) and oxidized flavoproteins (containing flavin adenine dinucleotide, i.e., FAD) has been demonstrated for potential biomedical applications including diagnosis, prognosis, and determining treatment response. However, the Chance redox scanner (a 3D cryogenic tissue imager) is limited by spatial resolution (~50 μm), and tissue ORI using fluorescence microscopy (single or multi-photon) is limited by the light penetration depth. Furthermore, viable or snap-frozen tissues are usually required. In this project, we aimed to study whether ORI may be achieved for unstained fixed tissue using a state-of-the-art modern Serial Two-Photon (STP) Tomography scanner that can rapidly acquire multi-plane images at micron resolution. Tissue specimens of mouse muscle, liver, and tumor xenografts were harvested and fixed in 4% parafor-maldehyde (PFA) for 24 h. Tissue blocks were scanned by STP Tomography under room temperature to acquire the autofluorescence signals (NADH channel: excitation 750 nm, blue emission filter; FAD channel: excitation 860 nm, green emission filter). We observed remarkable signals with significant intra-tissue heterogeneity in images of NADH, FAD and redox ratio (FAD/(NADH+FAD)), which are worthy of further investigation for extracting biological information.

Despite various technological advancements in cancer diagnosis, the mortality rates were not decreased significantly. We aim to develop a novel optical imaging tool to assist cancer diagnosis effectively. Fluorescence spectroscopy/imaging is a fast, rapid, and minimally invasive technique which has been successfully applied to diagnosing cancerous cells/tissues. Recently, the ratiometric imaging of intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), as pioneered by Britton Chance and the co-workers in 1950-70’s, has gained much attention to quantify the physiological parameters of living cells/tissues. The redox ratio, i.e., FAD/(FAD+NADH) or FAD/NADH, has been shown to be sensitive to various metabolic changes in in vivo and in vitro cells/tissues. Optical redox imaging has also been investigated for providing potential imaging biomarkers for cancer transformation, aggressiveness, and treatment response. Towards this goal, we have designed and developed a novel fiberoptic-based needle redox imager (NRI) that can fit into an 11G clinical coaxial biopsy needle for real time imaging during clinical cancer surgery. In the present study, the device is calibrated with tissue mimicking phantoms of FAD and NADH along with various technical parameters such as sensitivity, dynamic range, linearity, and spatial resolution of the system. We also conducted preliminary imaging of tissues ex vivo for validation. We plan to test the NRI on clinical breast cancer patients. Once validated this device may provide an effective tool for clinical cancer diagnosis.

We previously showed that optical redox imaging (ORI) of snap-frozen breast biopsies by the Chance redox scanner readily discriminates cancer from normal tissue. Moreover, indices of redox heterogeneity differentiate among tumor xenografts with different metastatic potential. These observations suggest that ORI of fluorescence of NADH and oxidized flavoproteins (Fp) may provide diagnostic/prognostic value for clinical applications. In this work, we investigate whether ORI of formalin-fixed-paraffin-embedded (FFPE) unstained clinical tissue slides of breast tumors is feasible and comparable to ORI of snap-frozen tumors. If ORI of FFPE is validated, it will enhance the versatility of ORI as a novel diagnostic/prognostic assay as FFPE samples are readily available. ORI of fixed tissue slides was performed using a fluorescence microscope equipped with a precision automated stage and appropriate optical filters. We developed a vignette correction algorithm to remove the tiling effect of stitched-images. The preliminary data from imaging fixed slides of breast tumor xenografts showed intratumor redox heterogeneity patterns similar to that of the frozen tissues imaged by the Chance redox scanner. From ORI of human breast tissue slides we identified certain redox differences among normal, ductal carcinoma in situ, and invasive carcinoma. We found paraformaldehyde fixation causes no change in NADH signals but enhances Fp signals of fresh muscle fibers. We also investigated the stability of the fluorescence microscope and reproducibility of tissue slide fluorescence signals. We plan to validate the diagnostic/prognostic value of ORI using clinically annotated breast cancer sample set from patients with long-term follow-up data.

Our previous studies indicate that the mitochondrial redox state and its intratumor heterogeneity are associated with invasiveness and metastatic potential in human breast cancer cell models and mouse xenografts. To further study the molecular basis of redox heterogeneity, we obtained the fluorescence images of Fp (oxidized flavoproteins containing flavin adenine dinucleotide, i.e., FAD), NADH (reduced nicotinamide adenine dinucleotide), and the Fp redox ratio (FpR = Fp/(Fp + NADH)) of MDA-MB-231 xenografts by the Chance redox scanner, then isolated the intratumoral redox subpopulations by dissection according to the redox ratio image. A total of 12 subpopulations were isolated from 4 tumors (2–4 locations from each tumor). The 12 subpopulations were classified into 3 FpR groups: high FpR (HFpR, n = 4, FpR range 0.78–0.92, average 0.85), medium FpR (MFpR, n = 5, FpR range 0.39–0.68, average 0.52), and low FpR (LFpR, n = 3, FpR range 0.15–0.28, average 0.20). The RT-PCR (reverse transcription polymerase chain reaction) analysis on these redox subpopulations showed that PGC-1α is significantly upregulated in the HFpR redox group compared to the MFpR group (fold change 2.1, p = 0.008), but not significantly different between MFpR and LFpR groups, or between HFpR and LFpR groups. These results indicate that optical redox imaging (ORI)-based redox subpopulations exhibit differential expression of PGC1α gene and suggest that PGC1α might play a role in redox mediation of breast cancer progression.

The anti-inflammatory genes, haem oxygenase 1 (HO-1, HMOX1) rs2071746 (unrestricted model: p = 9.07 × 10-4; recessive model: p = 4.99 × 10-4; multiplicative model: p = 0.0009; and additive model: p = 1.87 × 10-4) and interleukin-10 (IL-10) rs1800872 (dominant model: p = 0.0277) have been associated with paediatric inflammatory bowel disease. The present family-based case-trio study (n = 52) examined HO-1 gene expression in the presence of proinflammatory lipopolysaccharide and tumour necrosis factor-alpha in four B lymphocyte cell lines established from children with inflammatory bowel disease and demonstrated that mutations in IL-10 and IL-10 receptor B reduced HO-1 messenger RNA expression. This observation supports our hypothesis that HO-1 is regulated by the IL-10/STAT3 pathway and that both genes (IL10 and STAT3) could be involved in the pathogenesis of inflammatory bowel disease. We also compared HO-1 expression in diseased intestinal tissues with adjacent normal tissues from adults with inflammatory bowel disease. Of the 17 Crohn's disease patients, HO-1 expression in diseased tissues was downregulated in 9 patients (53%) and of the 10 ulcerative colitis patients HO-1 was downregulated in 7 patients (70%), compared with adjacent normal tissues. The downregulation of HO-1 gene expression may lower anti-inflammatory effects and worsen tissue injury in affected areas by inflammatory bowel disease.

Rationale and Objectives The clinical gold standard for breast cancer diagnosis relies on invasive biopsies followed by tissue fixation for subsequent histopathological examination. This process renders the specimens to be much less suitable for biochemical or metabolic analysis. Our previous metabolic imaging data in tumor xenograft models showed that the mitochondrial redox state is a sensitive indicator that can distinguish between normal and tumor tissue. In this study, we investigated whether the same redox imaging technique can be applied to core biopsy samples of human breast cancer and whether the mitochondrial redox state may serve as a novel metabolic biomarker that may be used to distinguish between normal and malignant breast tissue in the clinic. Our long-term objective was to identify novel metabolic imaging biomarkers for breast cancer diagnosis. Materials and Methods Both normal and cancerous tissue specimens were collected from the cancer-bearing breasts of three patients shortly after surgical resection. Core biopsies and tissue blocks were obtained from tumor and normal adjacent breast tissue, respectively. All specimens were snap-frozen with liquid nitrogen, embedded in chilled mounting medium with flavin adenine dinucleotide and reduced nicotinamide adenine dinucleotide reference standards adjacently placed, and scanned using the Chance redox scanner (ie, cryogenic nicotinamide adenine dinucleotide/oxidized flavoprotein fluorescence imager). Results Our preliminary data showed cancerous tissues had up to 10-fold higher oxidized flavoprotein signals and had elevated oxidized redox state compared to the normal tissues from the same patient. A high degree of tumor tissue heterogeneity in the redox indices was observed. Conclusions Our finding suggests that the identified redox imaging indices could differentiate between cancer and noncancer breast tissues without subjecting tissues to fixatives. We propose that this novel redox scanning procedure may assist in tissue diagnosis in freshly procured biopsy samples before tissue fixation.

Conventional methods for the analysis of in vivo hyperpolarized (13) C NMR data from the lactate dehydrogenase (LDH) reaction usually make assumptions on the stability of rate constants and/or the validity of the two-site exchange model. In this study, we developed a framework to test the validity of the assumption of stable reaction rate constants and the two-site exchange model in vivo via ratiometric fitting of the time courses of the signal ratio L(t)/P(t). Our analysis provided evidence that the LDH enzymatic kinetics observed by hyperpolarized NMR are in near-equilibrium and satisfy the two-site exchange model for only a specific time window. In addition, we quantified both the forward and reverse exchange rate constants of the LDH reaction for the transgenic and mouse xenograft models of breast cancer using the ratio fitting method developed, which includes only two modeling parameters and is less sensitive to the influence of instrument settings/protocols, such as flip angles, degree of polarization and tracer dosage. We further compared the ratio fitting method with a conventional two-site exchange modeling method, i.e. the differential equation fitting method, using both the experimental and simulated hyperpolarized NMR data. The ratio fitting method appeared to fit better than the differential equation fitting method for the reverse rate constant on the mouse tumor data, with less relative errors on average, whereas the differential equation fitting method also resulted in a negative reverse rate constant for one tumor. The simulation results indicated that the accuracy of both methods depends on the width of the transport function, noise level and rate constant ratio; one method may be more accurate than the other based on the experimental/biological conditions aforementioned. We were able to categorize our tumor models into specific conditions of the computer simulation and to estimate the errors of rate quantification. We also discussed possible approaches to the development of more accurate rate quantification methods for hyperpolarized NMR.

Our long-term goal was to investigate the potential of incorporating redox imaging technique as a breast cancer (BC) diagnosis component to increase the positive predictive value of suspicious imaging finding and to reduce unnecessary biopsies and overdiagnosis. We previously found that precancer and cancer tissues in animal models displayed abnormal mitochondrial redox state. We also revealed abnormal mitochondrial redox state in cancerous specimens from three BC patients. Here, we extend our study to include biopsies of 16 patients. Tissue aliquots were collected from both apparently normal and cancerous tissues from the affected cancer-bearing breasts shortly after surgical resection. All specimens were snap-frozen and scanned with the Chance redox scanner, i.e., the three-dimensional cryogenic NADH/Fp (reduced nicotinamide adenine dinucleotide/oxidized flavoproteins) fluorescence imager. We found both Fp and NADH in the cancerous tissues roughly tripled that in the normal tissues ( p < 0.05 ). The redox ratio Fp/(NADH + Fp) was ? 27 % higher in the cancerous tissues ( p < 0.05 ). Additionally, Fp, or NADH, or the redox ratio alone could predict cancer with reasonable sensitivity and specificity. Our findings suggest that the optical redox imaging technique can provide parameters independent of clinical factors for discriminating cancer from noncancer breast tissues in human patients.

Developing imaging biomarkers for non-invasive measurement of the tissue redox state is a key research area. Recently, we presented the first non-invasive MR imaging method that demonstrated the correlation between the endogenous chemical exchange saturation transfer (CEST) contrast and the tissue redox state. It is well known that the broadband magnetization transfer (MT) can occur via chemical exchange (CEST) and/or dipole–dipole interactions. The present study investigated if the broadband MT also correlated with the tissue redox state. The preliminary result for the prostate tumor xenografts indeed showed a significant correlation between the broadband MT contrast and the NADH redox ratio quantified with the optical redox scanning. In vivo MT contrast, once calibrated, may potentially serve as an imaging biomarker for tissue redox state.

Redox state mediates embryonic stem cell (ESC) differentiation and thus offers an important complementary approach to understanding the pluripotency of stem cells. NADH redox ratio (NADH/(Fp + NADH)), where NADH is the reduced form of nicotinamide adenine dinucleotide and Fp is the oxidized flavoproteins, has been established as a sensitive indicator of mitochondrial redox state. In this paper, we report our redox imaging data on the mitochondrial redox state of mouse ESC (mESC) colonies and the implications thereof. The low-temperature NADH/Fp redox scanner was employed to image mESC colonies grown on a feeder layer of gamma-irradiated mouse embryonic fibroblasts (MEFs) on glass cover slips. The result showed significant heterogeneity in the mitochondrial redox state within individual mESC colonies (size: ~200–440 μm), exhibiting a core with a more reduced state than the periphery. This more reduced state positively correlates with the expression pattern of Oct4, a well-established marker of pluripotency. Our observation is the first to show the heterogeneity in the mitochondrial redox state within a mESC colony, suggesting that mitochondrial redox state should be further investigated as a potential new biomarker for the stemness of embryonic stem cells.

We have imaged mitochondrial oxidation–reduction states by taking a ratio of mitochondrial fluorophores: NADH (reduced nicotinamide adenine dinucleotide) to Fp (oxidized flavoprotein). Although NADH has been investigated for tissue metabolic state in cancer and in oxygen deprived tissues, it alone is not an adequate measure of mitochondrial metabolic state since the NADH signal is altered by dependence on the number of mitochondria and by blood absorption. The redox ratio, NADH/(Fp + NADH), gives a more accurate measure of steady-state tissue metabolism since it is less dependent on mitochondrial number and it compensates effectively for hemodynamic changes. This ratio provides important diagnostic information in living tissues. In this study, the emitted fluorescence of mouse colon in situ is passed through an emission filter wheel and imaged on a CCD camera. Redox ratio images of the healthy and hypoxic mouse intestines clearly showed significant differences. Furthermore, the corrected redox ratio indicated an increase from an average value of 0.51 ± 0.10 in the healthy state to 0.92 ± 0.03 in dead tissue due to severe ischemia (N = 5). We show that the CCD imaging system is capable of displaying the metabolic differences in normal and ischemic tissues as well as quantifying the redox ratio in vivo as a marker of these changes.

An effective method for in vivo detection of early therapeutic response of patients with non-Hodgkin's lymphoma would enable personalized clinical management of cancer therapy and facilitate the design of optimal treatment regimens. This study evaluates the feasibility of T2-weighted MRI (T2WI) and diffusion-weighted MRI (DWI) for in vivo detection of response of human diffuse large B-cell lymphoma xenografts in severe combined immunodeficient mice to chemotherapy. Each cycle of combination chemotherapy with cyclophosphamide, hydroxydoxorubicin, Oncovin, prednisone, and bryostatin 1 (CHOPB) was administered to tumor-carrying mice weekly for up to four cycles. T2WI and DWI were performed before the initiation of CHOPB and after each cycle of CHOPB. In order to corroborate the MRI results, histological analyses were carried out on control tumors and treated tumors after completion of all MRI studies. DWI revealed a significant (P

Clinically translatable hyperpolarized (HP) (13)C-NMR can probe in vivo enzymatic reactions, e.g., lactate dehydrogenase (LDH)-catalyzed reaction by injecting HP (13)C-pyruvate into the subject, which is converted to (13)C labeled lactate by the enzyme. Parameters such as (13)C-lactate signals and lactate-to-pyruvate signal ratio are commonly used for analyzing the HP (13)C-NMR data. However, the biochemical/biological meaning of these parameters remains either unclear or dependent on experimental settings. It is preferable to quantify the reaction rate constants with a clearer physical meaning. Here we report the extraction of the kinetic parameters of the LDH reaction from HP (13)C-NMR data and investigate if they can be potential predictors of lung inflammation.Male Sprague-Dawley rats (12 controls, 14 treated) were used. One dose of bleomycin (2.5 U/kg) was administered intratracheally to the treatment group. The lungs were removed, perfused, and observed by the HP-NMR technique, where a HyperSense dynamic nuclear polarization system was used to generate the HP (13)C-pyruvate for injecting into the lungs. A 20 mm (1)H/(13)C dual-tuned coil in a 9.4-T Varian vertical bore NMR spectrometer was employed to acquire the (13)C spectral data every 1 s over a time period of 300 s using a non-selective, 15-degree radiofrequency pulse. The apparent rate constants of the LDH reaction and their ratio were quantified by applying ratiometric fitting analysis to the time series data of (13)C labeled pyruvate and lactate.The apparent forward rate constant kp =(3.67±3.31)×10(-4) s(-1), reverse rate constant kl =(4.95±2.90)×10(-2) s(-1), rate constant ratio kp /kl =(7.53±5.75)×10(-3) for the control lungs; kp =(11.71±4.35)×10(-4) s(-1), kl =(9.89±3.89)×10(-2) s(-1), and kp /kl =(12.39±4.18)×10(-3) for the inflamed lungs at the 7(th) day post treatment. Wilcoxon rank-sum test showed that the medians of these kinetic parameters of the 7-day cohort were significantly larger than those of the control cohort (P0.001, P=0.001, and P=0.019, respectively). The rate constants of individual lungs correlated significantly with the histology scores of neutrophils and organizing pneumonia foci but not macrophages. Both kp and kp /kl positively correlated with lactate labeling signals. No correlation was found between kl and lactate labeling signals.The results indicate bleomycin-induced lung inflammation significantly increased both the forward and reverse reaction rate constants of LDH and their ratio at day-7 after bleomycin treatment.

It has been shown that a malignant tumor is akin to a complex organ comprising of various cell populations including tumor cells that are genetically, metabolically and functionally different. Our redox imaging data have demonstrated intra-tumor redox heterogeneity in all mouse xenografts derived from human melanomas, breast, prostate, and colon cancers. Based on the signals of NADH and oxidized flavoproteins (Fp, including flavin adenine dinucleotide (FAD)) and their ratio, i.e., the redox ratio, which is an indicator of mitochondrial metabolic status, we have discovered several distinct redox subpopulations in xenografts of breast tumors potentially recapitulating functional/metabolic heterogeneity within the tumor. Furthermore, xenografts of breast tumors with higher metastatic potential tend to have a redox subpopulation whose redox ratio is significantly different from that of tumors with lower metastatic potential and usually have a bi-modal distribution of the redox ratio. The redox subpopulations from human breast cancer samples can also be very complex with multiple subpopulations as determined by fitting the redox ratio histograms with multi- Gaussian functions. In this report, we present a new method for identifying the redox subpopulations within individual breast tumor xenografts and human breast tissues, which may be used to differentiate between breast cancer and normal tissue and among breast cancer with different risks of progression.

The invasive/metastatic potential of cancer cells is an important factor in tumor progression. The redox ratios obtained from ratios of the endogenous fluorescent signals of NADH and FAD, can effectively respond to the alteration of cancer cells in its mitochondrial energy metabolism. It has been shown previously that the redox ratios may predict the metastatic potential of cancer mouse xenografts. In this report, we aimed to investigate the metabolic state represented by the redox ratios of cancer cells in vitro. Fluorescence microscopic imaging technology was used to observe the changes of the endogenous fluorescence signals of NADH and FAD in the energy metabolism pathways. We measured the redox ratios (FAD/NADH) of breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF-7, and SKBR3. We found that the more invasive cancer cells have higher FAD/NADH ratios, largely consistent with previous studies on breast cancer xenografts. Furthermore, by comparing the fluorescence signals of the breast cancer cells under different nutritional environments, including starvation and addition of glutamine, pyruvate and lactate, we found that the redox ratios still effectively distinguished the highly invasive MDA-MB-231 cells from less invasive MCF-7 cells. These preliminary data suggest that the redox ratio may potentially provide a new index to stratefy breast cancer with different degrees of aggressiveness, which could have significance for the diagnosis and treatment of breast cancer.

Abnormal metabolism can be a hallmark of cancer occurring ea rly before detectable histol ogical changes and may serve as an early detection biomarker. The current gold standard to establish breast cancer (BC) diagnosis is histological examination of biopsy. Previously we have found that pre-cancer and cancer tissues in animal models displayed abnormal mitochondrial redox state. Our technique of quantitatively measuring the mitochondrial redox state has the potential to be implemented as an early detection tool for cancer and may provide prognostic value. We therefore in this present study, investigated the feasibility of quantifying the redox state of tumor samples from 16 BC patients. Tumor tissue aliquots were collected from both normal and cancerous tissue from the affected cancer-bearing breasts of 16 female patients (5 TNBC, 9 ER

Tissue redox state is an important mediator of various biological processes in health and diseases such as cancer. Previously, we discovered that the mitochondrial redox state of ex vivo tissues detected by redox scanning (an optical imaging method) revealed interesting tumor redox state heterogeneity that could differentiate tumor aggressiveness. Because the noninvasive chemical exchange saturation transfer (CEST) MRI can probe the proton transfer and generate contrasts from endogenous metabolites, we aim to investigate if the in vivo CEST contrast is sensitive to proton transfer of the redox reactions so as to reveal the tissue redox states in breast cancer animal models.CEST MRI has been employed to characterize tumor metabolic heterogeneity and correlated with the redox states measured by the redox scanning in two human breast cancer mouse xenograft models, MDA-MB-231 and MCF-7. The possible biological mechanism on the correlation between the two imaging modalities was further investigated by phantom studies where the reductants and the oxidants of the representative redox reactions were measured.The CEST contrast is found linearly correlated with NADH concentration and the NADH redox ratio with high statistical significance, where NADH is the reduced form of nicotinamide adenine dinucleotide. The phantom studies showed that the reductants of the redox reactions have more CEST contrast than the corresponding oxidants, indicating that higher CEST effect corresponds to the more reduced redox state.This preliminary study suggests that CEST MRI, once calibrated, might provide a novel noninvasive imaging surrogate for the tissue redox state and a possible diagnostic biomarker for breast cancer in the clinic.

Rationale and Objectives Cancer cells generate more lactate than normal cells under both aerobic and hypoxic conditions—exhibiting the so-called Warburg effect. However, the relationship between the Warburg effect and tumor metastatic potential remains controversial. We intend to investigate whether the higher lactate reflects higher tumor metastatic potential. Materials and Methods We used hyperpolarized 13 C-pyruvate magnetic resonance spectroscopy (MRS) to compare lactate 13 C-labeling in vivo in mouse xenografts of the highly metastatic (MDA-MB-231) and the relatively indolent (MCF-7) human breast cancer cell lines. We obtained the kinetic parameters of the lactate dehydrogenase (LDH)–catalyzed reaction by three methods of data analysis including the differential equation fit, q-ratio fit, and ratio fit methods. Results Consistent results from the three methods showed that the highly metastatic tumors exhibited a smaller apparent forward rate constant ( k + = 0.060 ± 0.004 s −1 ) than the relatively indolent tumors ( k + = 0.097 ± 0.013 s −1 ). The ratio fit generated the greatest statistical significance for the difference ( P = .02). No significant difference in the reverse rate constant was found between the two tumor lines. Conclusions The result indicates that the less metastatic breast tumors may produce more lactate than the highly metastatic ones from the injected 13 C-pyruvate and supports the notion that breast tumor metastatic risk is not necessarily associated with the high levels of glycolysis and lactate production. More studies are needed to confirm whether and how much the measured apparent rate constants are affected by the membrane transporter activity and whether they are primarily determined by the LDH activity.