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.

Activation of P2X receptors induces apoptosis in human retinal pigment epithelium.

PURPOSE: The retinal pigment epithelium (RPE) is considered a primary site of pathology in age-related macular degeneration (AMD), which is the most prevalent form of irreversible blindness worldwide in the elderly population. Extracellular adenosine triphosphate (ATP) acts as a key signaling molecule in numerous cellular processes, including cell death. The purpose of this study was to determine whether extracellular ATP induces apoptosis in cultured human RPE. METHODS: RPE apoptosis was evaluated by caspase-3 activation, Hoechst staining, and DNA fragmentation. Intracellular Ca(2+) levels were determined by both a cell-based fluorometric Ca(2+) assay and a ratiometric Ca(2+) imaging technique. P2X(7) mRNA and protein expression were detected by reverse transcription-polymerase chain reaction (RT-PCR) and confocal microscopy, respectively. RESULTS: The authors found that both the endogenous P2X(7) agonist ATP and the synthetic, selective P2X(7) agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) induced RPE apoptosis, which was significantly inhibited by P2X(7) antagonist oxidized ATP (oATP) but not by the P2 receptor antagonist suramin; both ATP and BzATP increase intracellular Ca(2+) via extracellular Ca(2+) influx; both ATP- and BzATP-induced Ca(2+) responses were significantly inhibited by oATP but not by suramin; ATP-induced apoptosis was significantly inhibited or blocked by BAPTA-AM or by low or no extracellular Ca(2+); and P2X(7) receptor mRNA and protein were expressed in RPE cells. CONCLUSIONS: These findings suggest that P2X receptors, especially P2X(7) receptors, contribute to ATP- and BzATP-induced Ca(2+) signaling and apoptosis in the RPE. Abnormal Ca(2+) homeostasis through the activation of P2X receptors could cause the dysfunction and apoptosis of RPE that underlie AMD.

Calicum microdomains form within neutrophils at the neutrophil-tumor cell synapse: role in antibody-dependent target cell apoptosis.

Ca(2+) messages are broadly important in cellular signal transduction. In immune cells, Ca(2+) signaling is an essential step in many forms of activation. Neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is one form of leukocyte activation that plays an important role in tumor cell killing in vitro and in patient care. Using fluorescence methodologies, we found that neutrophils exhibit Ca(2+) signals during ADCC directed against breast fibrosarcoma cells. Importantly, these signals were localized to Ca(2+) microdomains at the neutrophil-to-tumor cell interface where they display dynamic features such as movement, fusion, and fission. These signals were blocked by the intracellular Ca(2+) buffer BAPTA. At the neutrophil-tumor cell synapse, the neutrophil's cytoplasm was enriched in STIM1, a crucial mediator of Ca(2+) signaling, whereas the Ca(2+)-binding proteins calbindin and parvalbumin were not affected. Our findings suggest that Ca(2+) microdomains are due to an active signaling process. As Ca(2+) signals within neutrophils were necessary for specific tumor cell apoptosis, a central role of microdomains in leukocyte-mediated tumor cell destruction is indicated.

Improved detection of nicotinamide adenine dinucleotide phosphate oscillations within human neutrophils.

Kinetic studies of nicotinamide adenine dinucleotide phosphate autofluorescence have been conducted in adherent neutrophils using an improved microscopic photometry system incorporating low noise excitation and detection systems. Dynamic autofluorescence oscillations were found with periods ranging from ∼4 min to ∼10 s. The largest portion of the population of oscillating neutrophils (32%) had periods near 2 min. The next largest group at 25% exhibited periods of 1 min or less. These oscillations could not be accounted for by instrument artifacts, cell shape changes away from the focal plane, or other factors. They disappeared when detergent was added to oscillating cells. Higher-frequency oscillations disappeared as cells changed shape, indicating a correlation between these two processes. This approach provides a reliable method to monitor this cellular property.

A cell permeant peptide containing the cytoplasmic tail sequence of Fc receptor type IIA reduces calcium signaling and phagolysosome formation in neutrophils.

Receptors for the Fc domain of IgG mediate target recognition, signal transduction, and effector functions including antibody-dependent cytolysis, phagocytosis, and phagolysosome formation. To better understand FcR-mediated functions and to identify potential therapeutic strategies, we employed cell-penetrating ("Trojan") peptides to deliver "wild-type" (LTL) or modified (AAA) FcgammaRIIA tail sequences to the neutrophil's cytoplasm. The Trojan-LTL peptide appeared to label the endoplasmic reticulum whereas the Trojan-AAA peptide distributed throughout the cytoplasm. The Trojan-LTL peptide, but not the Trojan-AAA peptide, decreased Ca(2+) signaling at the phagosome and reduced phagolysosome formation. These studies suggest that FcgammaRIIA's tail can act as a peptide decoy thereby blunting FcgammaRIIA-mediated processes, which, in turn, suggests a possible route in managing inflammatory tissue damage.

Observation of calcium microdomains at the uropod of living morphologically polarized human neutrophils using flash lamp-based fluorescence microscopy.

The present study outlines improved strategies for ratiometric imaging of cell calcium using a flash lamp-based excitation method and its application to neutrophil polarization. A brief (approximately 6 micros) and intense flash was used to excite the Fluo-4 and Fura Red calcium dye combination in morphologically polarized human neutrophils. These illumination conditions do not allow the dye or calcium ions to diffuse significant distances during the exposure period. Buffer conditions such as pH, pyruvate concentration, and glucose levels were adjusted to more faithfully replicate these parameters in sepsis patients. Fluorescence images at both dyes' emission wavelengths were simultaneously collected using a Dual-View apparatus and an ICCD camera. The ratiometric images, when viewed as single frames or averaged image stacks, clearly demonstrated high calcium probe ratios at the uropod and comparatively low ratios at the cell body that were not evident using conventional imaging methods with longer exposure times. Calcium signaling at the uropod is likely associated with cytoskeletal remodeling during cell motility.

The hexosamine biosynthesis pathway negatively regulates IL-2 production by Jurkat T cells.

To test the hypothesis that the hexosamine biosynthesis pathway (HBP) affects cytokine production, we studied IL-2 production by Jurkat cells in response to PHA. We found that the HBP activator glucosamine (GlcN), but not glucose (Glc), dose-dependently reduced IL-2 production. Importantly, GlcN blocked trafficking of a GFP-NFAT chimeric protein to the nucleus of stimulated transfectants. Not surprisingly, changes in O-GlcNAc protein modifications were noted during cell activation with and without GlcN addition. These findings could not be explained by some non-specific change in cell metabolism because ATP concentrations did not significantly change. We speculate that HBP-active compounds may contribute to patient care in certain inflammatory and autoimmune diseases.

Elevated glucose concentrations promote receptor-independent activation of adherent human neutrophils: an experimental and computational approach.

Neutrophil activation plays integral roles in host tissue damage and resistance to infectious diseases. As glucose uptake and NADPH availability are required for reactive oxygen metabolite production by neutrophils, we tested the hypothesis that pathological glucose levels (>or=12 mM) are sufficient to activate metabolism and reactive oxygen metabolite production in normal adherent neutrophils. We demonstrate that elevated glucose concentrations increase the neutrophil's metabolic oscillation frequency and hexose monophosphate shunt activity. In parallel, substantially increased rates of NO and superoxide formation were observed. However, these changes were not observed for sorbitol, a nonmetabolizable carbohydrate. Glucose transport appears to be important in this process as phloretin interferes with the glucose-specific receptor-independent activation of neutrophils. However, LY83583, an activator of glucose flux, promoted these changes at 1 mM glucose. The data suggest that at pathophysiologic concentrations, glucose uptake by mass action is sufficient to activate neutrophils, thus circumventing the normal receptor transduction mechanism. To enable us to mechanistically understand these dynamic metabolic changes, mathematical simulations were performed. A model for glycolysis in neutrophils was created. The results indicated that the frequency change in NAD(P)H oscillations can result from the activation of the hexose monophosphate shunt, which competes with glycolysis for glucose-6-phosphate. Experimental confirmation of these simulations was performed by measuring the effect of glucose concentrations on flavoprotein autofluorescence, an indicator of the rate of mitochondrial electron transport. Moreover, after prolonged exposure to elevated glucose levels, neutrophils return to a "nonactivated" phenotype and are refractile to immunologic stimulation. Our findings suggest that pathologic glucose levels promote the transient activation of neutrophils followed by the suppression of cell activity, which may contribute to nonspecific tissue damage and increased susceptibility to infections, respectively.

Myeloperoxidase accumulates at the neutrophil surface and enhances cell metabolism and oxidant release during pregnancy.

Pregnancy is a unique immunological state. Pregnancy neutrophils differ from those of non-pregnant women as they cannot be fully activated for oxidant production, but yet have higher levels of unstimulated oxidant production. Although reduced activation is due to decreased hexose monophosphate shunt activity, the mechanism enhancing basal oxidant levels is unknown. We hypothesize that myeloperoxidase (MPO) trafficking affects the basal oxidant release by maternal neutrophils. Immunofluorescence microscopy has demonstrated MPO at the surface of pregnancy neutrophils, whereas non-pregnancy cells do not exhibit surface MPO. Adherent pregnancy neutrophils were characterized by high-amplitude metabolic oscillations, which were blocked by MPO inactivation. Conversely, metabolic oscillatory amplitudes of control neutrophils were heightened by incubation with PMA or exogenous MPO. Importantly, MPO decoration of cell surfaces and high-amplitude metabolic oscillations were observed for neutrophils from pregnant but not from non-pregnant mice. However, cells from pregnant MPO knockout mice did not exhibit MPO expression or high-amplitude metabolic oscillations. Unstimulated neutrophils from pregnant women were found to release reactive oxygen metabolites (ROM) and reactive nitrogen intermediates (RNI), but cells from non-pregnant women did not. MPO inhibition returned ROM and RNI formation to non-pregnant levels. Hence, MPO trafficking influences metabolic activity and oxidant production in pregnancy.

Differential intracellular distributions of inositol trisphosphate and ryanodine receptors within and among hematopoietic cells.

To better understand the mechanism(s) of leukocyte Ca(2+) signaling, we have studied the intracellular locations of two Ca(2+)-mobilizing receptors, the inositol 1,4,5-trisphosphate receptor and ryanodine receptor, by immunofluorescence microscopy. Our results show that localization differs not only between receptor classes within a cell, but among leukocyte types as well. We also illustrate the importance of preserving labile cellular filaments in maintaining cell integrity by fixation with the Safiejko-Mroczka and Bell protocol, because conventional fixation methods distort receptor patterns. We suggest that the observed differences influence intracellular Ca(2+) signaling.

Identification of channels promoting calcium spikes and waves in HT1080 tumor cells: their apparent roles in cell motility and invasion.

Intracellular Ca(2+) signals have been associated with cell polarization and locomotion. As cell motility underlies metastasis, we have sought to better characterize the Ca(2+) signaling events in HT1080 fibrosarcoma cells. We have tested the hypothesis that low voltage-activated (LVA) and nonvoltage-gated (NVG) channels of HT1080 cells participate in dynamic Ca(2+)-signaling events leading to cell migration and invasion. Immunofluorescence microscopy has shown that HT1080 cells express LVA T-type Ca(2+) channels uniformly about the cell periphery, whereas the transient receptor potential-1 (a NVG cation channel) protein appears as punctate spots about a cell's periphery. HT1080 cells exhibit periodic intracellular Ca(2+) spikes. High-speed imaging revealed that the Ca(2+) spikes were composed of a single Ca(2+) wave traveling unidirectionally about the periphery of the cytoplasm in a clockwise fashion (as viewed from basal to apical surfaces). The T-type Ca(2+) channel blocker mibefradil inhibited Ca(2+) spikes and waves on cells and, in parallel, inhibited cell motility and invasion in a dose-dependent manner. Similar changes were noted with the NVG cation channel blockers Gd(3+) and carboxyamido-triazole. The combination of LVA and NVG blockers further reduced Matrigel invasiveness. However, the Ca(2+) channel blockers nicardipine, SKF96365, diltiazem, and verapamil had no effect at appropriate doses. These results indicate that certain LVA and NVG channels regulate HT1080 cell motility. In addition to providing novel information regarding cancer cell motility, we suggest that it may be possible to design drugs that inhibit a key Ca(2+) wave, thereby enhancing the efficacy of emerging therapeutic protocols.