flavin-adenine-dinucleotide and Breast-Neoplasms

Novel optoelectronic instrumentation has been developed for the multispectral imaging of autofluorescence emitted by metabolic fluorophores. The images resolve individual cells while spectra are collected for each pixel in the images. These datacubes are generated at a rate of 10 per second-fast enough for surgical guidance. The data is processed in real time to provide a single color-coded image to the surgeon. To date, the system has been applied to fresh, ex vivo, human surgical specimens and has distinguished breast cancer from benign tissue. The approach is applicable to in vivo measurements of surgical margins and needle-based optical biopsies. Ongoing work demonstrates that the system has great potential for translation to a hand-held probe with high sensitivity and specificity.

Topics: Biomarkers, Tumor; Biopsy; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; Luminescent Measurements; Margins of Excision; Mastectomy; NAD; Neoplasm, Residual; Optical Imaging; Predictive Value of Tests; Single-Cell Analysis

The enhanced uptake of glucose by cancer cells via aerobic glycolysis occurs when the lactic acid pathway is favored over the citric acid cycle. The lactic acid cycle in cancer cells influences the cytosolic concentration of metabolic fluorophores including NADH (the reduced form of nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD). In particular, the literature has shown that breast cancer influences the relative magnitude of fluorescence from NADH and FAD. A multispectral imaging system has been developed for rapid non-destructive imaging of intrinsic fluorescence in tissue. This paper compares in vivo data to fresh ex vivo data gathered as a function of time in mouse models. The data indicate that, if measured within 30 min of excision, a cancer diagnosis in fresh ex vivo tissue correlates with a cancer diagnosis in in vivo tissue. These results justify a plan to evaluate fresh ex vivo human tissue to quantify the sensitivity and specificity of the multispectral system.

Topics: Animals; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; Lactic Acid; Mice; NAD

This work aims to develop a clinically compatible system that can perform breast tissue analysis in a more time efficient process than conventional histopathological assessment. The potential for such a system to be used in vivo in the operating room or surgical suite to improve patient outcome is investigated.. In this work, 80 matched pairs of invasive ductal carcinoma and adjacent normal breast tissue were measured in a combined time-resolved fluorescence and diffuse reflectance (DA) system. Following measurement, the fluorescence intensity of collagen and flavin adenine dinucleotide (FAD); the fluorescence lifetime of collagen, nicotinamide adenine dinucleotide (NADH), and FAD; the DA; absorption coefficient; and reduced scattering coefficient were extracted. Samples then underwent histological processing and H&E staining to classify composition as tumor, fibroglandular, and/or adipose tissue.. Statistically significant differences in the collagen and FAD fluorescence intensity, collagen and FAD fluorescence lifetime, DA, and scattering coefficient were found between each tissue group. The NADH fluorescence lifetime and absorption coefficient were statistically different between the tumor and fibroglandular groups, and the tumor and adipose groups. While many breast tissue analysis studies label fibroglandular and adipose together as "normal" breast tissue, this work indicates that some differences between tumor and fibroglandular tissue are not the same as differences between tumor and adipose tissue. Observations of the reduced scatter coefficient may also indicate further classification to include fibro-adipose may be necessary. Future work would benefit from the additional tissue classification.. With observable differences in optical parameters between the three tissue types, this system shows promise as a breast analysis tool in a clinical setting. With further work involving samples of mixed composition, this combined system could potentially be used intraoperatively for rapid margin assessment.

Topics: Breast; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; NAD; Neoplasms; Spectrometry, Fluorescence

Flavin adenine dinucleotide (FAD) plays a key role in an extensive range of cellular oxidation-reduction reactions, which is engaged in metabolic pathways. The purpose of this study was to realize pegylated flavins formulation, named FAD and FAD-PEG diacid complex as theranostic tool in cancer therapy. For this objective, a murine breast cancer model, which was induced by mouse-derived4T1 breast cancer cells was studied to assess the therapeutic efficacy of FAD (named NP1) and FAD-PEG diacid complex (named NP2). The cytokines were monitored to evaluate the serum inflammatory factors to develop the blood cell content of different groups of nude mice. The experimental model shows that an intravenous injection of FAD (NP1) can significantly reduce tumour volume, tumour index and thymus index, and decrease neutrophils (NE), monocytes (MO), eosinophils (EO), and basophils (BA). At the same time, the content of IL-1α, IL-12P70, TNF α, IL-1β and IL-6 was significantly reduced, and the content of IL-10 was significantly increased. These results provide the proof-of-concept for FAD as a smart adjuvant for cancer therapy and encourages their further development in the field of Nanomedicine.

Topics: Animals; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; Mice; Mice, Nude; Polyethylene Glycols; Precision Medicine

Drug-resistant cells and anti-inflammatory immune cells within tumor masses contribute to tumor aggression, invasion, and worse patient outcomes. These cells can be a small proportion (<10%) of the total cell population of the tumor. Due to their small quantity, the identification of rare cells is challenging with traditional assays. Single cell analysis of autofluorescence images provides a live-cell assay to quantify cellular heterogeneity. Fluorescence intensities and lifetimes of the metabolic coenzymes reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide allow quantification of cellular metabolism and provide features for classification of cells with different metabolic phenotypes. In this study, Gaussian distribution modeling and machine learning classification algorithms are used for the identification of rare cells within simulated autofluorescence lifetime image data of a large tumor comprised of tumor cells and T cells. A Random Forest machine learning algorithm achieved an overall accuracy of 95% for the identification of cell type from the simulated optical metabolic imaging data of a heterogeneous tumor of 20,000 cells consisting of 70% drug responsive breast cancer cells, 5% drug resistant breast cancer cells, 20% quiescent T cells and 5% activated T cells. High resolution imaging methods combined with single-cell quantitative analyses allows identification and quantification of rare populations of cells within heterogeneous cultures.

Topics: Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; NAD; NADP; Optical Imaging

Extracellular vesicles (EVs) shuttle proteins, RNA, DNA, and lipids crucial for cell-to-cell communication. Recent findings have highlighted that EVs, by virtue of their cargo, may also contribute to breast cancer (BC) growth and metastatic dissemination. Indeed, EVs are gaining great interest as non-invasive cancer biomarkers. However, little is known about the biological and physical properties of EVs from malignant BC lesions, and even less is understood about EVs from non-malignant lesions, such as breast fibroadenoma (FAD), which are clinically managed using conservative approaches. Thus, for this pilot study, we attempted to purify and explore the proteomic profiles of EVs from benign breast lesions, HER2+ BCs, triple-negative BCs (TNBCs), and continuous BC cell lines (i.e., BT-549, MCF-10A, and MDA-MB-231), combining experimental and semi-quantitative approaches. Of note, proteome-wide analyses showed 49 common proteins across EVs harvested from FAD, HER2+ BCs, TNBCs, and model BC lines. This is the first feasibility study evaluating the physicochemical composition and proteome of EVs from benign breast cells and primary and immortalized BC cells. Our preliminary results hold promise for possible implications in precision medicine for BC.

Topics: Breast Neoplasms; Cell Line, Tumor; Extracellular Vesicles; Female; Fibroadenoma; Flavin-Adenine Dinucleotide; Humans; Pilot Projects; Proteome; Proteomics

Macrophages within the tumor microenvironment (TME) exhibit a spectrum of protumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate two-photon autofluorescence imaging of NAD(P)H and FAD to nondestructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours poststimulation for mouse macrophages (RAW264.7) stimulated with IFNγ or IL4 plus IL13 in 2D culture, confirming that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse polyoma-middle T virus or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor cocultures exhibited significantly different FAD mean lifetimes and greater migration than monocultures at 24, 48, and 72 hours postseeding. In cocultures with primary human cancer cells, actively migrating monocyte-derived macrophages had greater redox ratios [NAD(P)H/FAD intensity] compared with passively migrating monocytes at 24 and 48 hours postseeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumor-immune cross-talk within the 3D TME. SIGNIFICANCE: Label-free metabolic imaging and microscale culture technologies enable monitoring of single-cell macrophage metabolism, migration, and function in the 3D tumor microenvironment.

Topics: Animals; Breast Neoplasms; Cell Culture Techniques; Cell Movement; Coculture Techniques; Female; Flavin-Adenine Dinucleotide; Humans; Imaging, Three-Dimensional; Lab-On-A-Chip Devices; Macrophages; Mice; NADP; Optical Imaging; RAW 264.7 Cells; Tumor Microenvironment

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.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Heterografts; Humans; Image Processing, Computer-Assisted; Mice; NAD; Optical Imaging; Oxidation-Reduction; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha

Clinical cancer treatment aims to target all cell subpopulations within a tumor. Autofluorescence microscopy of the metabolic cofactors NAD(P)H and FAD has shown sensitivity to anti-cancer treatment response. Alternatively, flow cytometry is attractive for high throughput analysis and flow sorting. This study measures cellular autofluorescence in three flow cytometry channels and applies cellular autofluorescence to sort a heterogeneous mixture of breast cancer cells into subpopulations enriched for each phenotype. Sorted cells were grown in culture and sorting was validated by morphology, autofluorescence microscopy, and receptor expression. Ultimately, this method could be applied to improve drug development and personalized treatment planning.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Separation; Flavin-Adenine Dinucleotide; Flow Cytometry; Fluorescence; Humans; NADP

Macrophage infiltration and recruitment in breast tumors has been correlated with poor prognosis in breast cancer patients and has been linked to tumor cell dissemination. Much of our understanding comes from animal models in which macrophages are labeled by expression of an extrinsic fluorophore. However, conventional extrinsic fluorescence labeling approaches are not readily applied to human tissue and clinical use. We report a novel strategy that exploits endogenous fluorescence from the metabolic co-factors NADH and FAD with quantitation from Fluorescence Lifetime Imaging Microscopy (FLIM) as a means to non-invasively identify tumor-associated macrophages in the intact mammary tumor microenvironment. Macrophages were FAD(HI) and demonstrated a glycolytic-like NADH-FLIM signature that was readily separated from the intrinsic fluorescence signature of tumor cells. This non-invasive quantitative technique provides a unique ability to discern specific cell types based upon their metabolic signatures without the use of exogenous fluorescent labels. Not only does this provide high resolution temporal and spatial views of macrophages in live animal breast cancer models, this approach can be extended to other animal disease models where macrophages are implicated and has potential for clinical applications.

Topics: Animals; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; Macrophages; Microscopy, Fluorescence; NAD; Optical Imaging

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.

Topics: Biomarkers, Tumor; Breast Neoplasms; Cell Movement; Energy Metabolism; Female; Flavin-Adenine Dinucleotide; Humans; MCF-7 Cells; Microscopy, Fluorescence; Mitochondria; NAD; Neoplasm Invasiveness; Oxidation-Reduction; Rotenone; Tumor Microenvironment; Uncoupling Agents

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.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Breast; Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Humans; Image Interpretation, Computer-Assisted; Middle Aged; Mitochondria; Molecular Imaging; NAD; Oxidation-Reduction; ROC Curve

There is a need for accurate, high-throughput, functional measures to gauge the efficacy of potential drugs in living cells. As an early marker of drug response in cells, cellular metabolism provides an attractive platform for high-throughput drug testing. Optical techniques can noninvasively monitor NADH and FAD, two autofluorescent metabolic coenzymes. The autofluorescent redox ratio, defined as the autofluorescence intensity of NADH divided by that of FAD, quantifies relative rates of cellular glycolysis and oxidative phosphorylation. However, current microscopy methods for redox ratio quantification are time-intensive and low-throughput, limiting their practicality in drug screening. Alternatively, high-throughput commercial microplate readers quickly measure fluorescence intensities for hundreds of wells. This study found that a commercial microplate reader can differentiate the receptor status of breast cancer cell lines (p < 0.05) based on redox ratio measurements without extrinsic contrast agents. Furthermore, microplate reader redox ratio measurements resolve response (p < 0.05) and lack of response (p > 0.05) in cell lines that are responsive and nonresponsive, respectively, to the breast cancer drug trastuzumab. These studies indicate that the microplate readers can be used to measure the redox ratio in a high-throughput manner and are sensitive enough to detect differences in cellular metabolism that are consistent with microscopy results.

Topics: Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Female; Flavin-Adenine Dinucleotide; High-Throughput Screening Assays; Humans; NAD; Optical Imaging; Oxidation-Reduction

Abnormal cellular metabolism is a hallmark of cancer, yet there is an absence of quantitative methods to dynamically image this powerful cellular function. Optical metabolic imaging (OMI) is a noninvasive, high-resolution, quantitative tool for monitoring cellular metabolism. OMI probes the fluorescence intensities and lifetimes of the autofluorescent metabolic coenzymes reduced NADH and flavin adenine dinucleotide. We confirm that OMI correlates with cellular glycolytic levels across a panel of human breast cell lines using standard assays of cellular rates of glucose uptake and lactate secretion (P < 0.05, r = 0.89). In addition, OMI resolves differences in the basal metabolic activity of untransformed from malignant breast cells (P < 0.05) and between breast cancer subtypes (P < 0.05), defined by estrogen receptor and/or HER2 expression or absence. In vivo OMI is sensitive to metabolic changes induced by inhibition of HER2 with the antibody trastuzumab (herceptin) in HER2-overexpressing human breast cancer xenografts in mice. This response was confirmed with tumor growth curves and stains for Ki67 and cleaved caspase-3. OMI resolved trastuzumab-induced changes in cellular metabolism in vivo as early as 48 hours posttreatment (P < 0.05), whereas fluorodeoxyglucose-positron emission tomography did not resolve any changes with trastuzumab up to 12 days posttreatment (P > 0.05). In addition, OMI resolved cellular subpopulations of differing response in vivo that are critical for investigating drug resistance mechanisms. Importantly, OMI endpoints remained unchanged with trastuzumab treatment in trastuzumab-resistant xenografts (P > 0.05). OMI has significant implications for rapid cellular-level assessment of metabolic response to molecular expression and drug action, which would greatly accelerate drug development studies.

Topics: Animals; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Disease Models, Animal; Female; Flavin-Adenine Dinucleotide; Glycolysis; Humans; Mice; Mice, Nude; NAD; Optical Imaging; Receptor, ErbB-2; Trastuzumab; Xenograft Model Antitumor Assays

Cowden syndrome (CS), a Mendelian autosomal-dominant disorder, predisposes to breast, thyroid and other cancers. Germline mutations in phosphatase and tensin homolog (PTEN) have been recently reported in 23% of a large series of classic CS. Here, we validated our small (n = 10) pilot study in a large patient series that germline variations in succinate dehydrogenase genes (SDHx) occur in 8% (49/608) of PTEN mutation-negative CS and CS-like (CSL) individuals (SDH(var+)). None of these SDHx variants was found in 700 population controls (P < 0.0001). We then found that SDHx variants also occur in 6% (26/444) of PTEN mutation-positive (PTEN(mut+)) CS/CSL individuals (PTEN(mut+)/SDH(var+)). Of 22 PTEN(mut+)/SDH(var+) females, 17 had breast cancers compared with 34/105 PTEN(mut+) (P < 0.001) or 27/47 SDH(var+) patients (P = 0.06). Notably, individuals with SDH(var+) alone had the highest thyroid cancer prevalence (24/47) compared with PTEN(mut+) patients (27/105, P = 0.002) or PTEN(mut+)/SDH(var+) carriers (6/22, P = 0.038). Patient-derived SDH(var+) lymphoblastoid cells had elevated cellular reactive oxygen species, highest in PTEN(mut+)/SDH(var+) cells, correlating with apoptosis resistance. SDH(var+) cells showed stabilized and hyperactivated hypoxia inducible factor (HIF)1α signaling. Most interestingly, we also observed the loss of steady-state p53 in the majority of SDH(var+) cells. This loss of p53 was regulated by MDM2-independent NADH quinone oxidoreductase 1-mediated protein degradation, likely due to the imbalance of flavin adenine dinucleotide/nicotinamide adenine dinucleotide in SDH(var+) cells. Our data suggest the potential regulation of HIF1α, p53 and PTEN signaling by mitochondrial metabolism in CS/CSL tumorigenesis. Together, our findings suggest the importance of considering SDHx as candidate predisposing and modifier genes for CS/CSL-related malignancy risks, and a mechanism which suggests ways of therapeutic reversal or prevention.

Topics: Breast Neoplasms; Female; Flavin-Adenine Dinucleotide; Genes, p53; Genetic Carrier Screening; Genetic Predisposition to Disease; Germ-Line Mutation; Hamartoma Syndrome, Multiple; Humans; NAD; PTEN Phosphohydrolase; Succinate Dehydrogenase; Thyroid Neoplasms

Stokes Shift (SS) Spectroscopy (SSS) of normal and abnormal breast and prostate tissues were studied. SS spectra is measured by simultaneously scanning both the excitation and emission wavelengths while keeping a fixed wavelength interval of Δλ = 20 nm. Characteristic, highly resolved peaks and significant spectral differences between normal and different pathological tissues of breast and prostate tissues were observed. The SS spectra of normal and different pathological breast and prostate tissues show the distinct peaks around 300, 350, 450, 500 and 600 nm may be attributed to tryptophan, collagen, NADH, flavin and porphyrin, respectively. Results of the current study demonstrate that the SS spectral changes due to tryptophan, collagen, hemoglobin, NADH, FAD and porphyrin have good diagnostic potential; therefore can be targeted as native tumor markers.

Topics: Adult; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Fibroadenoma; Flavin-Adenine Dinucleotide; Humans; Male; Middle Aged; NAD; Pilot Projects; Porphyrins; Prostatic Hyperplasia; Prostatic Neoplasms; Spectrometry, Fluorescence; Spectrophotometry; Young Adult

Autofluorescence spectroscopy is a powerful imaging technique that exploits endogenous fluorophores. The endogenous fluorophores NADH and flavin adenine dinucleotide (FAD) are two of the principal electron donors and acceptors in cellular metabolism, respectively. The optical oxidation-reduction (redox) ratio is a measure of cellular metabolism and can be determined by the ratio of NADH/FAD. We hypothesized that there would be a significant difference in the optical redox ratio of normal mammary epithelial cells compared with breast tumor cell lines and that estrogen receptor (ER)-positive cells would have a higher redox ratio than ER-negative cells. To test our hypothesis, the optical redox ratio was determined by collecting the fluorescence emission for NADH and FAD via confocal microscopy. We observed a statistically significant increase in the optical redox ratio of cancer compared with normal cell lines (P < 0.05). Additionally, we observed a statistically significant increase in the optical redox ratio of ER(+) breast cancer cell lines. The level of ESR1 expression, determined by real-time PCR, directly correlated with the optical redox ratio (Pearson's correlation coefficient = 0.8122, P = 0.0024). Furthermore, treatment with tamoxifen and ICI 182,870 statistically decreased the optical redox ratio of only ER(+) breast cancer cell lines. The results of this study raise the important possibility that fluorescence spectroscopy can be used to identify subtypes of breast cancer based on receptor status, monitor response to therapy, or potentially predict response to therapy. This source of optical contrast could be a potentially useful tool for drug screening in preclinical models.

Topics: Breast Neoplasms; Cell Line, Tumor; Epithelial Cells; Estradiol; Estrogen Receptor alpha; Estrogen Receptor Modulators; Female; Flavin-Adenine Dinucleotide; Fulvestrant; Humans; Mammary Glands, Human; Microscopy, Confocal; NAD; Oxidation-Reduction; RNA, Messenger; Tamoxifen

The classical examination of histology slides from a mouse model of breast cancer has been extended in this study to incorporate modern multiphoton excitation and photon-counting techniques. The advantage of such approaches is quantification of potential diagnostic parameters from the fluorescence emission signal, whereby the traditional descriptive staging process is complemented by measurements of fluorescence intensity, lifetime, and spectra. We explored whether the clinical "gold standard" of eosin and hematoxylin stained histology slides would provide optical biomarker signatures of diagnostic value. Alternatively, we examined unstained slides for changes in intensity and/or fluorescence lifetime of relevant endogenous fluorophores. Although eosin provided a strong emission signal and had distinct spectra and lifetime, we found that it was not useful as a fluorescent biological marker, particularly when combined with hematoxylin. Instead, we found that the properties of the fluorescence from the endogenous fluorophores NADH and FAD were indicative of the pathological state of the tissue. Comparing regions of carcinoma in situ to adjacent histologically normal regions, we found that tumor cells produced higher intensity and had a longer fluorescence lifetime. By imaging at 780 nm and 890 nm excitation, we were able to differentiate the fluorescence of FAD from NADH by separating the emission spectra. The shift to a longer lifetime in tumor cells was independent of the free or bound state of FAD and NADH, and of the excitation wavelength. Most forms of cancer have altered metabolism and redox ratios; here we present a method that has potential for early detection of these changes, which are preserved in fixed tissue samples such as classic histopathology slides.

Topics: Animals; Biomarkers; Breast; Breast Neoplasms; Carcinoma in Situ; Cell Transformation, Neoplastic; Eosine Yellowish-(YS); Epithelial Cells; Epithelium; Flavin-Adenine Dinucleotide; Fluorescence; Hematoxylin; Humans; Mice; Microscopy, Fluorescence, Multiphoton; NAD; Staining and Labeling

Photobleaching and recovery of 488-nm excited fluorescence from resected human breast tissue samples have been studied. Profiles of photobleaching decay were seen to be faster in cancerous tissue than in those of the normal tissue. The reverse behavior was observed in profiles of recovery after photobleaching. A theoretical model based on one-dimensional diffusion theory has been developed to provide insight into the phenomena of fluorescence during photobleaching and recovery in a multiply scattering medium such as tissue. To understand photobleaching and recovery with the help of this theoretical model, we carried out experiments with model media that were prepared with authentic fluorophores, scatterers, and absorbers. The results of these studies suggest that the fluorescence photobleaching profiles are affected more by the absorption than by the scattering properties of a turbid medium such as tissue. In contrast, the scattering properties of the medium are found to affect the fluorescence recovery profiles to a greater extent. These observations could be related to the observed difference in fluorescence photobleaching and recovery profiles of normal and cancerous breast tissues.

Topics: Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Diffusion; Female; Flavin-Adenine Dinucleotide; Fluorescence Recovery After Photobleaching; Humans; Models, Theoretical; Phantoms, Imaging; Protoporphyrins

Opioids and nitric oxide (NO) interact functionally in different systems. NO-generating agents decrease the activity of opioid agonists, prevent opioid tolerance, and are used in opioid withdrawal syndromes. There exist, however, few reports indicating a direct interaction of the two systems. T47D human breast cancer cells in culture express opioid receptors, and opioid agonists inhibit their growth, while they release high amounts of the NO-related molecules NO(2-)/NO(3-)to the culture medium. We have used this system to assay a possible direct interaction of opiergic and nitric oxide systems. Our results show that delta- or mu-acting opioid agonists do not modify the release of NO(2-)/NO(3-). In contrast, kappa-acting opioid agonists (ethylketocyclazocine, and alpha(S1)-casomorphine) decrease the release of NO(2-)/NO(3-), in a time- and dose-dependent manner. The general opioid antagonist diprenorphine (10(-6) M) produce a similar NO(2-)/NO(3-)release inhibition, indicating a possible non-opioid-receptor mediated phenomenon. In addition, ethylketocyclazocine, alpha(S1)-casomorphin and diprenorphine directly inhibit NOS activity: agonists, interact with both calcium-dependent and independent NOS-isoforms, while the antagonist diprenorphine modifies only the activity of the calcium-dependent fraction of the enzyme. Analysis of this interaction revealed that opioids modify the dimeric active form of NOS, through binding to the reductase part of the molecule, acting as non-competitive inhibitors of the enzyme. This interaction opens interesting new possibilities for tumor biology and breast cancer therapy.

Topics: Breast Neoplasms; Calcium; Culture Media, Conditioned; Dose-Response Relationship, Drug; Enzyme Activation; Flavin-Adenine Dinucleotide; Humans; Isoenzymes; Kinetics; Narcotic Antagonists; Narcotics; Nitric Oxide; Nitric Oxide Synthase; Substrate Specificity; Tumor Cells, Cultured

Flavoprotein reductases play a key role in electron transfer in many physiological processes. We have isolated a cDNA with strong sequence similarities to cytochrome P-450 reductase and nitric-oxide synthase. The cDNA encodes a protein of 597 amino acid residues with a predicted molecular mass of 67 kDa. Northern blot analysis identified a predicted transcript of 3.0 kilobase pairs as well as a larger transcript at 6.0 kilobase pairs, and the gene was mapped to chromosome 9q34.3 by fluorescence in situ hybridization analysis. The amino acid sequence of the protein contained distinct FMN-, FAD-, and NADPH-binding domains, and in order to establish whether the protein contained these cofactors, the coding sequence was expressed in insect cells and purified. Recombinant protein bound FMN, FAD, and NADPH cofactors and exhibited a UV-visible spectrum with absorbance maxima at 380, 460, and 626 nm. The purified enzyme reduced cytochrome c, with apparent K(m) and k(cat) values of 21 microM and 1.3 s(-1), respectively, and metabolized the one-electron acceptors doxorubicin, menadione, and potassium ferricyanide. Immunoblot analysis of fractionated MCF7 cells with antibodies to recombinant NR1 showed that the enzyme is cytoplasmic and highly expressed in a panel of human cancer cell lines, thus indicating that this novel reductase may play a role in the metabolic activation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Breast Neoplasms; Chromosome Mapping; Chromosomes, Human, Pair 6; Cloning, Molecular; Cytochromes c1; Female; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; FMN Reductase; HeLa Cells; Humans; Kinetics; Mice; Molecular Sequence Data; Molecular Weight; NADH, NADPH Oxidoreductases; NADP; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Spectrophotometry; Tumor Cells, Cultured