A CLIA/CAP Compliant Noninvasive Laboratory Developed Test for Early Detection of Pancreatic Ductal Adenocarcinoma.

A noninvasive test for earlier detection of pancreatic cancer in individuals at higher risk is currently unavailable. To fill this void, we devised PancSure, a laboratory developed test in compliance with clinical regulations. PancSure is based on the protein biomarkers LYVE1 and REG1B, measured in urine by enzyme-linked immunosorbent assay, and commonly utilized serum/plasma CA19.

Evaluation of urinary C-reactive protein as an early detection biomarker for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide. Up to now, no specific screening or diagnostic tests are available for early PDAC detection. As a result, most patients are diagnosed with advanced or metastatic disease, which leads to a poor prognosis.

Highly sensitive noninvasive early lung cancer detection using DNA methylation topology in sputum-derived epithelial cells.

Objective: Sputum is a source of exfoliated respiratory epithelial cells transformed early in lung carcinogenesis. Malignant cells are hypomethylated and contain less genomic 5-methylcytosine (5mC). Validating a test that recognizes and quantifies aberrantly hypomethylated cells in sputum, we assessed its potential as a screening tool for detecting early-stage non-small cell lung cancer.

DNA methylation topology differentiates between normal and malignant in cell models, resected human tissues, and exfoliated sputum cells of lung epithelium.

Background: Global DNA hypomethylation is a prominent feature of cancer cells including lung cancer, that has not been widely explored towards cancer diagnosis. In this study we assess the comparative distribution of global DNA methylation in normal cells versus cancer cells in various specimen models.

Methods: We used immunofluorescence labeling of overall 5-methylcytosine (5mC) and covisualization of global DNA (gDNA) by 4',6-diamidino-2-phenylindole (DAPI), confocal microscopy and 3D image analysis to derive 5mC/DAPI colocalization patterns in human cell lines (BEAS-2B, A549, H157) and upper respiratory epithelial cells derived from various sources (i.

Covisualization of Global DNA Methylation/Hydroxymethylation and Protein Biomarkers for Ultrahigh-Definition Epigenetic Phenotyping of Stem Cells.

DNA methylation and DNA hydroxymethylation are genomic-scale key regulatory modifications in cellular differentiation and are skewed in complex diseases. Therefore, analyzing the nuclear distribution of globally methylated and hydroxymethylated DNA in conjunction with relevant cellular components, such as protein biomarkers, may well add cell-by-cell-specific spatial and temporal information to quantitative molecular data for the discovery of signaling networks in stem cell differentiation and their exploitation in the therapeutic reprogramming of cells. Fluorescence imaging provides an optical approach that has become an essential tool in this context.

An Inhibitor of GSK3B and HDACs Kills Pancreatic Cancer Cells and Slows Pancreatic Tumor Growth and Metastasis in Mice.

Background & Aims: Growth, progression, and drug resistance of pancreatic ductal adenocarcinomas (PDACs) have been associated with increased levels and activity of glycogen synthase kinase 3 beta (GSK3B) and histone deacetylases (HDACs). We designed and synthesized molecules that simultaneously inhibit the activities of both enzymes. We tested the effects of one of these molecules, Metavert, in pancreatic cancer cells and mice with pancreatic tumors.

Prostate cancer diagnosis using epigenetic biomarkers, 3D high-content imaging and probabilistic cell-by-cell classifiers.

Background: Prostate cancer (PCa) management can benefit from novel concepts/biomarkers for reducing the current 20-30% chance of false-negative diagnosis with standard histopathology of biopsied tissue.

Method: We explored the potential of selected epigenetic markers in combination with validated histopathological markers, 3D high-content imaging, cell-by-cell analysis, and probabilistic classification in generating novel detailed maps of biomarker heterogeneity in patient tissues, and PCa diagnosis. We used consecutive biopsies/radical prostatectomies from five patients for building a database of ∼140,000 analyzed cells across all tissue compartments and for model development; and from five patients and the two well-characterized HPrEpiC primary and LNCaP cancer cell types for model validation.

Rapid 3-D delineation of cell nuclei for high-content screening platforms.

High-resolution three-dimensional (3-D) microscopy combined with multiplexing of fluorescent labels allows high-content analysis of large numbers of cell nuclei. The full automation of 3-D screening platforms necessitates image processing algorithms that can accurately and robustly delineate nuclei in images with little to no human intervention. Imaging-based high-content screening was originally developed as a powerful tool for drug discovery.

Dynamic heterogeneity of DNA methylation and hydroxymethylation in embryonic stem cell populations captured by single-cell 3D high-content analysis.

Unlabelled: Cell-surface markers and transcription factors are being used in the assessment of stem cell fate and therapeutic safety, but display significant variability in stem cell cultures. We assessed nuclear patterns of 5-hydroxymethylcytosine (5hmC, associated with pluripotency), a second important epigenetic mark, and its combination with 5-methylcytosine (5mC, associated with differentiation), also in comparison to more established markers of pluripotency (Oct-4) and endodermal differentiation (FoxA2, Sox17) in mouse embryonic stem cells (mESC) over a 10-day differentiation course in vitro: by means of confocal and super-resolution imaging together with 3D high-content analysis, an essential tool in single-cell screening.

In Summary: 1) We did not measure any significant correlation of putative markers with global 5mC or 5hmC.

Covisualization of methylcytosine, global DNA, and protein biomarkers for In Situ 3D DNA methylation phenotyping of stem cells.

DNA methylation and histone modifications are key regulatory mechanisms in cellular differentiation, and are skewed in complex diseases. Therefore, analyzing the higher nuclear organization of methylated DNA in conjunction with relevant cellular components, such as protein biomarkers, may well add cell-by-cell-specific spatial and temporal information to quantitative molecular data for the discovery of stem cell differentiation-related signaling networks and their exploitation in the therapeutic reprogramming of cells. The in situ fluorescent covisualization of methylated DNA (methylated CG dinucleotides = MeC), global DNA (gDNA), and proteins has been challenging, as the immunofluorescence detection of MeC sites requires thorough denaturing of double-stranded DNA for antigen (methylated carbon-5 of cytosine) retrieval.

Nuclear DNA methylation and chromatin condensation phenotypes are distinct between normally proliferating/aging, rapidly growing/immortal, and senescent cells.

This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns - visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis - in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development.

3-D DNA methylation phenotypes correlate with cytotoxicity levels in prostate and liver cancer cell models.

Background: The spatial organization of the genome is being evaluated as a novel indicator of toxicity in conjunction with drug-induced global DNA hypomethylation and concurrent chromatin reorganization. 3D quantitative DNA methylation imaging (3D-qDMI) was applied as a cell-by-cell high-throughput approach to investigate this matter by assessing genome topology through represented immunofluorescent nuclear distribution patterns of 5-methylcytosine (MeC) and global DNA (4,6-diamidino-2-phenylindole = DAPI) in labeled nuclei.

Methods: Differential progression of global DNA hypomethylation was studied by comparatively dosing zebularine (ZEB) and 5-azacytidine (AZA).

DNA methylation topology: potential of a chromatin landmark for epigenetic drug toxicology.

Targeting chromatin and its basic components through epigenetic drug therapy has become an increased focus in the treatment of complex diseases. This boost calls for the implementation of high-throughput cell-based assays that exploit the increasing knowledge about epigenetic mechanisms and their interventions for genotoxicity testing of epigenetic drugs. 3D quantitative DNA methylation imaging is a novel approach for detecting drug-induced DNA demethylation and concurrent heterochromatin decondensation/reorganization in cells through the analysis of differential nuclear distribution patterns of methylcytosine and gDNA visualized by fluorescence and processed by machine-learning algorithms.

Early in vitro differentiation of mouse definitive endoderm is not correlated with progressive maturation of nuclear DNA methylation patterns.

The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6), and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells.

Measuring topology of low-intensity DNA methylation sites for high-throughput assessment of epigenetic drug-induced effects in cancer cells.

Epigenetic anti-cancer drugs with demethylating effects have shown to alter genome organization in mammalian cell nuclei. The interest in the development of novel epigenetic drugs has increased the demand for cell-based assays to evaluate drug performance in pre-clinical studies. An imaging-based cytometrical approach that can measure demethylation effects as changes in the spatial nuclear distributions of methylated cytosine and global DNA in cancer cells is introduced in this paper.

Automated quantification of DNA demethylation effects in cells via 3D mapping of nuclear signatures and population homogeneity assessment.

Today's advanced microscopic imaging applies to the preclinical stages of drug discovery that employ high-throughput and high-content three-dimensional (3D) analysis of cells to more efficiently screen candidate compounds. Drug efficacy can be assessed by measuring response homogeneity to treatment within a cell population. In this study, topologically quantified nuclear patterns of methylated cytosine and global nuclear DNA are utilized as signatures of cellular response to the treatment of cultured cells with the demethylating anti-cancer agents: 5-azacytidine (5-AZA) and octreotide (OCT).

Proteinase and growth factor alterations revealed by gene microarray analysis of human diabetic corneas.

Purpose: To identify proteinases and growth factors abnormally expressed in human corneas of donors with diabetic retinopathy (DR), additional to previously described matrix metalloproteinase (MMP)-10 and -3 and insulin-like growth factor (IGF)-I.

Methods: RNA was isolated from 35 normal, diabetic, and DR autopsy human corneas ex vivo or after organ culture. Amplified cRNA was analyzed using 22,000-gene microarrays (Agilent Technologies, Palo Alto, CA).

Topology of genes and nontranscribed sequences in human interphase nuclei.

Knowledge about the functional impact of the topological organization of DNA sequences within interphase chromosome territories is still sparse. Of the few analyzed single copy genomic DNA sequences, the majority had been found to localize preferentially at the chromosome periphery or to loop out from chromosome territories. By means of dual-color fluorescence in situ hybridization (FISH), immunolabeling, confocal microscopy, and three-dimensional (3D) image analysis, we analyzed the intraterritorial and nuclear localization of 10 genomic fragments of different sequence classes in four different human cell types.

Evaluation of techniques using amplified nucleic acid probes for gene expression profiling.

Gene expression analyses using spotted cDNA microarrays typically require relatively large quantities of total RNA (up to 100 microg) or polyA+RNA (1-5 microg). However, samples obtained by microdissection, patient biopsies, or embryonic samples often are small and yield an insufficient amount of RNA. Methods such as linear RNA amplification by in vitro transcription (IVT) or cDNA amplification by PCR are currently being used to circumvent these limitations.