flavin-adenine-dinucleotide and Precancerous-Conditions

While metabolic changes are considered a cancer hallmark, their assessment has not been incorporated in the detection of early or precancers, when treatment is most effective. Here, we demonstrate that metabolic changes are detected in freshly excised human cervical precancerous tissues using label-free, non-destructive imaging of the entire epithelium. The images rely on two-photon excited fluorescence from two metabolic co-enzymes, NAD(P)H and FAD, and have micron-level resolution, enabling sensitive assessments of the redox ratio and mitochondrial fragmentation, which yield metrics of metabolic function and heterogeneity. Simultaneous characterization of morphological features, such as the depth-dependent variation of the nuclear:cytoplasmic ratio, is demonstrated. Multi-parametric analysis combining several metabolic metrics with morphological ones enhances significantly the diagnostic accuracy of identifying high-grade squamous intraepithelial lesions. Our results motivate the translation of such functional metabolic imaging to

Topics: Cervix Uteri; Epithelium; Female; Flavin-Adenine Dinucleotide; Humans; Metabolic Networks and Pathways; Mitochondrial Dynamics; NAD; NADP; Optical Imaging; Precancerous Conditions; Reproducibility of Results; Sensitivity and Specificity; Staining and Labeling; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Multi-photon fluorescence microscopy techniques allow for non-invasive interrogation of live samples in their native environment. These methods are particularly appealing for identifying pre-cancers because they are sensitive to the early changes that occur on the microscopic scale and can provide additional information not available using conventional screening techniques.. In this study, we developed novel automated approaches, which can be employed for the real-time analysis of two-photon fluorescence images, to non-invasively discriminate between normal and pre-cancerous/HPV-immortalized engineered tissues by concurrently assessing metabolic activity, morphology, organization, and keratin localization. Specifically, we found that the metabolic activity was significantly enhanced and more uniform throughout the depths of the HPV-immortalized epithelia, based on our extraction of the NADH and FAD fluorescence contributions. Furthermore, we were able to separate the keratin contribution from metabolic enzymes to improve the redox estimates and to use the keratin localization as a means to discriminate between tissue types. To assess morphology and organization, Fourier-based, power spectral density (PSD) approaches were employed. The nuclear size distribution throughout the epithelial depths was quantified by evaluating the variance of the corresponding spatial frequencies, which was found to be greater in the normal tissue compared to the HPV-immortalized tissues. The PSD was also used to calculate the Hurst parameter to identify the level of organization in the tissues, assuming a fractal model for the fluorescence intensity fluctuations within a field. We found the range of organization was greater in the normal tissue and closely related to the level of differentiation.. A wealth of complementary morphological, biochemical and organizational tissue parameters can be extracted from high resolution images that are acquired based entirely on endogenous sources of contrast. They are promising diagnostic parameters for the non-invasive identification of early cancerous changes and could improve significantly diagnosis and treatment for numerous patients.

Topics: Cell Line; Cells, Cultured; Flavin-Adenine Dinucleotide; Humans; Keratinocytes; Keratins; Microscopy, Fluorescence, Multiphoton; NAD; Precancerous Conditions; Tissue Engineering

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

Topics: Animals; Carcinoma; Cell Nucleus; Cricetinae; Cytoplasm; Flavin-Adenine Dinucleotide; Fluorescence; Microscopy, Fluorescence, Multiphoton; Mouth Neoplasms; NAD; Oxidation-Reduction; Precancerous Conditions; Tumor Cells, Cultured

Topics: Carcinoma in Situ; Female; Flavin-Adenine Dinucleotide; Humans; NAD; Precancerous Conditions; Spectrometry, Fluorescence; Uterine Cervical Neoplasms