Computer Vision Identifies Recurrent and Nonrecurrent Ductal Carcinoma in Situ Lesions with Special Emphasis on African-American Women.

Although nonrecurrent and recurrent forms of ductal carcinoma in situ (DCIS) of the breast are observed, no evidence-based test can make this distinction. The current retrospective case-control study used archival DCIS samples stained with anti-phospho-Ser226-glucose transporter type 1 and anti-phosphofructokinase type L antibodies. Immunofluorescence micrographs were used to create machine learning models of recurrent and nonrecurrent biomarker patterns, which were evaluated in cross-validation studies. Clinical performance was assessed by holdout studies using patients whose data were not used in training. Micrographs were stratified according to the recurrence probability of each image. Recurrent patients were defined by at least one image with a probability of recurrence ≥98%, whereas nonrecurrent patients had none. These studies found no false-negatives, identified true-positives, and uniquely identified true-negatives. Roughly 20% of the microscope fields of recurrent lesions were computationally recurrent. Strong prognostic results were obtained for both white and African-American women. This machine tool provides the first means to accurately predict recurrent and nonrecurrent patient outcomes. Data indicate that at least some false-positive findings were true-positive findings that benefited from surgical intervention. The intracellular locations of phospho-Ser226-glucose transporter type 1 and phosphofructokinase type L likely participate in cancer recurrences by accelerating glucose flux, a key feature of the Warburg effect.

Enzyme trafficking and coclustering precede and accurately predict human breast cancer recurrences: an interdisciplinary review.

Although great effort has been expended to understand cancer's origins, less attention has been given to the primary cause of cancer deaths-cancer recurrences and their sequelae. This interdisciplinary review addresses mechanistic features of aggressive cancer by studying metabolic enzyme patterns within ductal carcinoma in situ (DCIS) of the breast lesions. DCIS lesions from patients who did or did not experience a breast cancer recurrence were compared. Several proteins, including phospho-Ser226-glucose transporter type 1, phosphofructokinase type L and phosphofructokinase/fructose 2,6-bisphosphatase type 4 are found in nucleoli of ductal epithelial cells in samples from patients who will not subsequently recur, but traffic to the cell periphery in samples from patients who will experience a cancer recurrence. Large coclusters of enzymes near plasmalemmata will enhance product formation because enzyme concentrations in clusters are very high while solvent molecules and solutes diffuse through small channels. These structural changes will accelerate aerobic glycolysis. Agglomerations of pentose phosphate pathway and glutathione synthesis enzymes enhance GSH formation. As aggressive cancer lesions are incomplete at early stages, they may be unrecognizable. We have found that machine learning provides superior analyses of tissue images and may be used to identify biomarker patterns associated with recurrent and nonrecurrent patients with high accuracy. This suggests a new prognostic test to predict patients with DCIS who are likely to recur and those who are at low risk for recurrence. Mechanistic interpretations provide a deeper understanding of anticancer drug action and suggest that aggressive metastatic cancer cells are sensitive to reductive chemotherapy.

Using Machine Vision of Glycolytic Elements to Predict Breast Cancer Recurrences: Design and Implementation.

A major goal of biomedical research has been the early and quantitative identification of patients who will subsequently experience a cancer recurrence. In this review, I discuss the ability of glycolytic enzyme and transporter patterns within tissues to detect sub-populations of cells within ductal carcinoma in situ (DCIS) lesions that specifically precede cancer recurrences. The test uses conventional formalin fixed paraffin embedded tissue samples. The accuracy of this machine vision test rests on the identification of relevant glycolytic components that promote enhanced glycolysis (phospho-Ser226-glucose transporter type 1 (phospho-Ser226-GLUT1) and phosphofructokinase type L (PFKL)), their trafficking in tumor cells and tissues as judged by computer vision, and their high signal-to-noise levels. For each patient, machine vision stratifies micrographs from each lesion as the probability that the lesion originated from a recurrent sample. This stratification method removes overlap between the predicted recurrent and non-recurrent patients, which eliminates distribution-dependent false positives and false negatives. The method identifies computationally negative samples as non-recurrent and computationally positive samples are recurrent; computationally positive non-recurrent samples are likely due to mastectomies. The early phosphorylation and isoform switching events, spatial locations and clustering constitute important steps in metabolic reprogramming. This work also illuminates mechanistic steps occurring prior to a recurrence, which may contribute to the development of new drugs.

Relocation of phosphofructokinases within epithelial cells is a novel event preceding breast cancer recurrence that accurately predicts patient outcomes.

Although recurrent cancers can become life threatening, little is known about the intracellular events required for cancer recurrences. Due to this lack of mechanistic information, there is no test to predict cancer recurrences or nonrecurrences during early stages of disease. In this retrospective study, we use ductal carcinoma in situ of the breast as a framework to better understand the mechanism of cancer recurrences using patient outcomes as the physiological observable. Conventional pathology slides were labeled with anti-phosphofructokinase type L (PFKL) and anti-phosphofructokinase/fructose-2,6-bisphosphatase type 4 (PFKFB4) reagents. PFKL and PFKFB4 were found in ductal epithelial cell nucleoli from DCIS samples of women who did not experience a cancer recurrence. In contrast, PFKL and PFKFB4 may be found near the plasma membrane in samples from patients who will develop recurrent cancer. With the use of machine learning to predict patient outcomes, holdout studies of individual patient micrographs for the three biomarkers PFKL, PFKFB4, and phosphorylated glucose transporter 1 (GLUT1) demonstrated 38.6% true negatives, 49.5% true positives, 11.9% false positives, and 0% false negatives (n = 101). A subpopulation of recurrent samples demonstrated PFKL, PFKFB4, and phosphorylated GLUT1 accumulation at the apical surface of epithelial cells, suggesting that carbohydrates can be harvested from the ducts' luminal spaces as an energy source. We suggest that PFK isotype patterns are metabolic switches representing key mechanistic steps of recurrences. Furthermore, PFK enzyme patterns within epithelial cells contribute to an accurate diagnostic test to classify DCIS patients as high or low recurrence risk.

Spatial locations of certain enzymes and transporters within preinvasive ductal epithelial cells predict human breast cancer recurrences.

Some patients treated for ductal carcinoma in situ (DCIS) of the breast will experience cancer recurrences, whereas other patients will not. Unfortunately, current techniques cannot identify which preinvasive lesions will lead to recurrent cancer. Because the mechanism of cancer recurrence is unknown, it is difficult to design a test that detects its activity. We propose that certain pentose phosphate pathway enzymes, glutathione synthesis enzymes, and RhoA cluster at the epithelial cell periphery before cancer recurrences. Enzyme clustering enhances metabolic flux. Using fluorescence microscopy, we show that phosphophorylated glucose transporter type-1, transketolase-like protein-1, glutathione synthetase, GTP-loaded RhoA, and RhoA accumulate as a peripheral layer near the epithelial cell surface in surgical biopsies of women who will suffer recurrences, but not in samples from women who will not experience recurrences as judged using 2×2 contingency tables. Machine-learning studies of phospho-glucose transporter type 1-labeled tissue sections of patients with DCIS demonstrated strong cross-validation and holdout performance. A machine study of individual cribriform, papillary, micropapillary, and comedo forms of DCIS demonstrated 97% precision and 95% recall in the detection of samples from women who will not experience a recurrence and 90% precision and 94% recall in the detection of lesions that will become recurrent. A holdout study of these patients showed 73% true negatives, 18% true positives, 4% false positives, and 4% false negatives at a 50% threshold. This work suggests mechanistic features of cancer recurrences that may contribute to a new clinical test distinguishing high from low-recurrence risk in patients with DCIS.

Frontiers of Complex Disease Mechanisms: Membrane Surface Tension May Link Genotype to Phenotype in Glaucoma.

Although many monogenic diseases are understood based upon structural changes of gene products, less progress has been made concerning polygenic disease mechanisms. This article presents a new interdisciplinary approach to understand complex diseases, especially their genetic polymorphisms. I focus upon primary open angle glaucoma (POAG). Although elevated intraocular pressure (IOP) and oxidative stress are glaucoma hallmarks, the linkages between these factors and cell death are obscure. Reactive oxygen species (ROS) promote the formation of oxidatively truncated phosphoglycerides (OTP), free fatty acids, lysophosphoglycerides, oxysterols, and other chemical species that promote membrane disruption and decrease membrane surface tension. Several POAG-linked gene polymorphisms identify proteins that manage damaged lipids and/or influence membrane surface tension. POAG-related genes expected to participate in these processes include: ELOVL5, ABCA1, APOE4, GST, CYP46A1, MYOC, and CAV. POAG-related gene products are expected to influence membrane surface tension, strength, and repair. I propose that heightened IOP overcomes retinal ganglion cell (RGC) membrane compressive strength, weakened by damaged lipid accumulation, to form pores. The ensuing structural failure promotes apoptosis and blindness. The linkage between glaucoma genotype and phenotype is mediated by physical events. Force balancing between the IOP and compressive strength regulates pore nucleation; force balancing between pore line tension and membrane surface tension regulates pore growth. Similar events may contribute to traumatic brain injury, Alzheimer's disease, and macular degeneration.

Protocol for Biomarker Ratio Imaging Microscopy with Specific Application to Ductal Carcinoma In situ of the Breast.

This protocol describes the methods and steps involved in performing biomarker ratio imaging microscopy (BRIM) using formalin fixed paraffin-embedded (FFPE) samples of human breast tissue. The technique is based on the acquisition of two fluorescence images of the same microscopic field using two biomarkers and immunohistochemical tools. The biomarkers are selected such that one biomarker correlates with breast cancer aggressiveness while the second biomarker anti-correlates with aggressiveness. When the former image is divided by the latter image, a computed ratio image is formed that reflects the aggressiveness of tumor cells while increasing contrast and eliminating path-length and other artifacts from the image. For example, the aggressiveness of epithelial cells may be assessed by computing ratio images of N-cadherin and E-cadherin images or CD44 and CD24 images, which specifically reflect the mesenchymal or stem cell nature of the constituent cells, respectively. This methodology is illustrated for tissue samples of ductal carcinoma in situ (DCIS) and invasive breast cancer. This tool should be useful in tissue studies of experimental cancer as well as the management of cancer patients.

Identification of lesion subtypes in biopsies of ductal carcinoma in situ of the breast using biomarker ratio imaging microscopy.

Although epidemiological studies propose aggressive and non-aggressive forms of ductal carcinoma in situ (DCIS), they cannot be identified with conventional histopathology. We now report a retrospective study of human biopsy samples using biomarker ratio imaging microscopy (BRIM). Using BRIM, micrographs of biomarkers whose expression correlates with breast cancer aggressiveness are divided by micrographs of biomarkers whose expression negatively correlates with aggressiveness to create computed micrographs reflecting aggressiveness. The biomarker pairs CD44/CD24, N-cadherin/E-cadherin, and CD74/CD59 stratified DCIS samples. BRIM identified subpopulations of DCIS lesions with ratiometric properties resembling either benign fibroadenoma or invasive carcinoma samples. Our work confirms the existence of distinct subpopulations of DCIS lesions, which will likely have utility in breast cancer research and clinical practice.

WO3/Pt nanoparticles promote light-induced lipid peroxidation and lysosomal instability within tumor cells.

Although metal-metal oxide nanoparticles have attracted considerable interest as catalysts, they have attracted little interest in nanomedicine. This is likely due to the fact that metal oxide semiconductors generally require biologically harmful ultraviolet excitation. In contrast, this study focuses upon WO3/Pt nanoparticles, which can be excited by visible light. To optimize the nanoparticles' catalytic performance, platinization was performed at alkaline pH. These nanoparticles destroyed organic dyes, consumed dissolved oxygen and produced hydroxyl radicals. 4T1 breast cancer cells internalized WO3/Pt nanoparticles within the membrane-bound endo-lysosomal compartment as shown by electron and fluorescence microscopy. During visible light exposure, but not in darkness, WO3/Pt nanoparticles manufacture reactive oxygen species, promote lipid peroxidation, and trigger lysosomal membrane disruption. As cells of the immune system degrade organic molecules, produce reactive oxygen species, and activate the lipid peroxidation pathway within target cells, these nanoparticles mimic the chemical attributes of immune effector cells. These biomimetic nanoparticles should become useful in managing certain cancers, especially ocular cancer.

WO3/Pt nanoparticles are NADPH oxidase biomimetics that mimic effector cells in vitro and in vivo.

To provide a means of delivering an artificial immune effector cell-like attack on tumor cells, we report the tumoricidal ability of inorganic WO3/Pt nanoparticles that mimic a leukocyte's functional abilities. These nanoparticles route electrons from organic structures and electron carriers to form hydroxyl radicals within tumor cells. During visible light exposure, WO3/Pt nanoparticles manufacture hydroxyl radicals, degrade organic compounds, use NADPH, trigger lipid peroxidation, promote lysosomal membrane disruption, promote the loss of reduced glutathione, and activate apoptosis. In a model of advanced breast cancer metastasis to the eye's anterior chamber, we show that WO3/Pt nanoparticles prolong the survival of 4T1 tumor-bearing Balb/c mice. This new generation of inorganic photosensitizers do not photobleach, and therefore should provide an important therapeutic advance in photodynamic therapy. As biomimetic nanoparticles destroy targeted cells, they may be useful in treating ocular and other forms of cancer.

Titanium-doped cerium oxide nanoparticles protect cells from hydrogen peroxide-induced apoptosis.

To develop new nanoparticle materials possessing anti-oxidative capacity with improved physical characteristics, we have studied titanium-doped cerium oxide (CeTiO2) nanoparticles. CeTiO2 nanoparticles had a mode diameter of 15-20 nm. These nanoparticles demonstrated catalase activity, and did not promote the activation of hemolytic or cytolytic pathways in living cells. Using surface plasmon resonance enhanced microscopy, we find that these nanoparticles associate with cells. Transmission electron microscopy studies demonstrated that these nanoparticles accumulate within the vacuolar compartment of cells. Importantly, CeTiO2 nanoparticles decrease hydrogen peroxide-mediated apoptosis of cells as judged by the reduced cleavage of a caspase 3-sensitive label. CeTiO2 nanoparticles may contribute to deflecting tissue damage in a broad spectrum of oxidant-mediated diseases, such as macular degeneration and Alzheimer's disease.

Noninvasive imaging of mitochondrial dysfunction in dry age-related macular degeneration.

BACKGROUND AND OBJECTIVE: Oxidative stress and mitochondrial dysfunction are implicated in the pathogenesis of age-related macular degeneration (AMD). Because increased flavoprotein fluorescence (FPF) is indicative of mitochondrial dysfunction, the authors attempted to detect mitochondrial dysfunction in eyes with AMD using FPF. PATIENTS AND METHODS: Six nonexudative eyes with AMD, including three with geographic atrophy (GA), and age-matched control eyes were imaged with a FPF device. Qualitative and quantitative analyses were conducted on the FPF images. RESULTS: Five eyes with AMD, including all three eyes with GA, showed qualitative and/or quantitative FPF heterogeneity that was not present in control eyes. Mean FPF average intensities of eyes with AMD with (P = .044) and without (P = .00060) GA were significantly greater than those of control eyes. The standard deviations of FPF images were greater in eyes with AMD (P = .020). CONCLUSION: In this small cluster of patients with AMD, retinal FPF is increased, suggesting elevated mitochondrial dysfunction. FPF heterogeneity indicates that an increased variability in mitochondrial dysfunction seems to be present in eyes with advanced disease.

Titanium doping reduces superoxide dismutase activity, but not oxidase activity, of catalytic CeO(2) nanoparticles.

In this paper we report the enzymatic properties of Ti-doped CeO(2) nanoparticles. The superoxide dismutase activity of Ti-doped nanoparticles is reduced in comparison to undoped nanoceria. In contrast, the oxidase activity of these nanoparticles was unchanged. The change in enzymatic activity was accompanied by a dramatic change in shape to a spherical nanostructure. In addition to reporting a new type of enzymatically-active nanoparticle, Ti-doped cerium oxide nanoparticles are well suited for biological applications.

Cerium oxide and platinum nanoparticles protect cells from oxidant-mediated apoptosis.

Catalytic nanoparticles represent a potential clinical approach to replace or correct aberrant enzymatic activities in patients. Several diseases, including many blinding eye diseases, are promoted by excessive oxidant stress due to reactive oxygen species (ROS). Cerium oxide and platinum nanoparticles represent two potentially therapeutic nanoparticles that de-toxify ROS. In the present study we directly compare these two classes of catalytic nanoparticles. Cerium oxide and platinum nanoparticles were found to be 16±2.4 nm and 1.9±0.2 nm in diameter, respectively. Using surface plasmon enhanced microscopy, we find that these nanoparticles associate with cells. Furthermore, cerium oxide and platinum nanoparticles demonstrated superoxide dismutase catalytic activity, but did not promote hemolytic or cytolytic pathways in living cells. Importantly, both cerium oxide and platinum nanoparticles reduce oxidant-mediated apoptosis in target cells as judged by the activation of caspase 3. The ability to diminish apoptosis may contribute to maintaining healthy tissues.

An enzymatic colorimetric assay for glucose-6-phosphate.

A specific colorimetric assay for the determination of glucose-6-phosphate (G6P) was developed. This assay is based on the oxidation of G6P in the presence of glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP(+)); the NADPH thereby generated reduces the tetrazolium salt WST-1 [2-(4-indophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt] to water-soluble yellow-colored formazan with 1-methoxy-5-methylphenazium methylsulfate (1-mPMS) as an electron carrier. The assay is optimized for reaction buffer pH, enzyme/dye concentration, and reaction time course. The limit of detection of the assay is 0.15 µM (15 pmol/well). The usefulness of the assay is demonstrated by the accurate measurement of the G6P concentration in fetal bovine serum (FBS).

Retinal flavoprotein fluorescence correlates with mitochondrial stress, apoptosis, and chemokine expression.

Oxidative stress and mitochondrial dysfunction occur before apoptosis in many retinal diseases. Under these conditions, a larger fraction of flavoproteins become oxidized and, when excited by blue-light, emit green flavoprotein fluorescence (FPF). In this study, we evaluated the utility of FPF as an early indicator of mitochondrial stress, pre-apoptotic cellular instability, and apoptosis of human retinal pigment epithelial (HRPE) cells subjected to hydrogen peroxide (H(2)O(2)) or monocytes (unstimulated or interferon-γ-stimulated) in vitro and of freshly-isolated pieces of human and rat neural retina subjected to H(2)O(2)ex vivo. Increased FPF of HRPE cells exposed to H(2)O(2) correlated with reduced mitochondrial membrane potential (ΔΨm) and increased apoptosis in a time- and dose-dependent manner. HRPE cells co-cultured with monocytes had increased FPF that correlated in a time-dependent manner with reduced ΔΨm, increased apoptosis, and early expression of pro-inflammatory chemokines, interleukin-8 (IL8) and monocyte chemotactic factor-1 (MCP1), which are known to be induced by oxidative stress. Increased FPF, reduced ΔΨm, and upregulation of IL8 and MCP1 occurred as early as 1-2 h after exposure to stressors, while apoptosis did not occur in HRPE cells until later time points. The antioxidant, N-acetyl-cysteine (NAC), inhibited increased FPF and apoptosis of HRPE cells subjected to H(2)O(2). Increased FPF of human and rat neural retina also correlated with increased apoptosis. This study suggests that FPF is a useful measure of mitochondrial function in retinal cells and tissues and can detect early mitochondrial dysfunction that may precede apoptosis.

Ischemia-induced nitrotyrosine formation and nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in human retinal pigment epithelium in vivo.

Reactive oxidative compounds including superoxide anions and nitric oxide are believed to play a central role in many blinding eye diseases. In the present study, we examine the effect of ischemia on human retinal pigment epithelial (RPE) cells in an unusual clinical case. We show that ischemia leads to extensive nitrotyrosine deposition in the RPE and choroid, thus indicating NO-dependent oxidative stress. We also show for the first time the in vivo translocation of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) to the nuclei of RPE cells. This enzyme's nuclear translocation has previously been demonstrated in vitro where it is a marker of apoptosis. Furthermore, nitrotyrosine deposition and GAPDH translocation have been duplicated in vitro using human RPE cells. Thus, nitrotyrosine formation and GAPDH trafficking to the nucleus may be observed during ischemic conditions.

MCP-1-activated monocytes induce apoptosis in human retinal pigment epithelium.

PURPOSE: The inflammatory response in age-related macular degeneration (AMD) is characterized by mononuclear leukocyte infiltration of the outer blood-retina barrier formed by the retinal pigment epithelium (RPE). A key mechanistic element in AMD progression is RPE dysfunction and apoptotic cell loss. The purpose of this study was to evaluate whether monocyte chemoattractant protein (MCP)-1-activated monocytes induce human RPE apoptosis and whether Ca(2+) and reactive oxygen species (ROS) are involved in this process. METHODS: A cell-based fluorometric assay was used to measure intracellular Ca(2+) concentrations ([Ca(2+)](i)) in RPE cells loaded with fluorescent Ca(2+) indicator. Intracellular RPE ROS levels were measured by using the 5- and 6-chloromethyl-2',7'-dichlorodihydrofluorescence diacetate acetyl ester (CM-H(2)DCFDA) assay. RPE apoptosis was evaluated by activated caspase-3, Hoechst staining, and apoptosis ELISA. RESULTS: MCP-1-activated human monocytes increased [Ca(2+)](i), ROS levels, and apoptosis in RPE cells, all of which were inhibited by 8-bromo-cyclic adenosine diphosphoribosyl ribose (8-Br-cADPR), an antagonist of cADPR. Although the ROS scavengers pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) significantly inhibited ROS production and apoptosis induced by activated monocytes, they did not affect induced Ca(2+) levels. The induced Ca(2+) levels and apoptosis in RPE cells were inhibited by an antibody against cluster of differentiation antigen 14 (CD14), an adhesion molecule expressed by these cells. CONCLUSIONS: These results indicate that CD14, Ca(2+), and ROS are involved in activated monocyte-induced RPE apoptosis and that cADPR contributes to these changes. Understanding the complex interactions among CD14, cADPR, Ca(2+), and ROS may provide new insights and treatments of retinal diseases, including AMD.