Name: imaging approaches to assessments of toxicological oxidative stress using genetically-encoded fluorogenic sensors
Uploaded: 2018-02-07T14:00:00
Description: Watch this Scientific Journal Video about Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors at JoVE.com

The authors would like to thank Katelyn Lavrich for assistance with experimental design and manuscript edits.

1. Preparation of Cells
NOTE: This procedure describes the lentiviral transduction of an immortalized cell line (BEAS-2B; ATCC, Manassas, VA) to express the desired reporter (roGFP2 or HyPer). Other cell lines/types and/or methods of gene transfer, including transfection, can be utilized as long as they result in a level of reporter expression adequate to visualize a sufficient number of sensor-expressing cells per field of view (typically 5 - 10 cells). If using transfection methods, the proced...

The use of roGFP2 and HyPer in detecting changes in intracellular EGSH and H2O2, respectively, has been well-described in previous physiological and toxicological studies 25,35,36,39,40,41,42,43. The protocol outlined here r...

The term &#34;oxidative stress&#34; is frequently cited as a mechanism in toxicology, yet rarely is this term described specifically. Oxidative stress can refer to several intracellular processes, including generation of reactive oxygen species, damage caused by free radicals, the oxidation of antioxidant molecules, and even the activation of specific signaling cascades. A broad range of environmental contaminants1,2 and pharmaceutical agents3,4 have been documented to induce oxidative stress by either direct action of the xenobiotic compound itself5,6 or secondarily by production of oxidant species as part of a cellular response7,8,9,10. It is therefore of great interest in toxicology to accurately observe and characterize the oxidative processes leading to adverse outcomes. Conventional methods of measuring oxidative stress involve identification of oxidized biomolecules11,12,13,14,15 or antioxidants16,17,18,19,20, or direct measurement of reactive species themselves21,22,23,24. However, these methods typically require cellular disruption, which often consumes the sample, eliminates spatial resolution, and potentially introduces artifacts25. The development of more sensitive and specific methods for the detection of oxidant species and markers of oxidative stress is broadly applicable to the investigation of the adverse effects of xenobiotic exposure.
Live-cell microscopy using a new generation of genetically-encoded fluorogenic sensors has emerged as a powerful tool to monitor the intracellular redox status of living cells. These sensors are typically expressed using a vector under the control of a viral promoter that is introduced via transfection or transduction methodologies. High expression efficiencies are not necessary, since cells expressing the fluorescent sensor can be easily identified visually. For toxicological assessments, cells expressing these sensors can be observed using fluorescence microscopy as they are exposed to xenobiotic compounds in real-time. This experimental design permits repeated measurements in the same cell, allowing each cell&#39;s established baseline to act as its own control. The high temporal resolution afforded by live-cell imaging is well-suited for the detection of oxidative events, particularly those that are modest in magnitude or transient in nature. In addition to being both sensitive and specific to their target molecules, the fluorescence of some of these sensors can be excited using two wavelengths of light. This phenomenon allows the fluorescent emission to be expressed as a ratio, which permits the discernment of signal changes associated with authentic sensor responses from those caused by artefacts such as variations in sensor expression, cell thickness, lamp intensity, photobleaching, and sensitivity of the fluorescence detector26. Another advantage of the use of fluorogenic sensors is that they can be targeted to specific cellular compartments, creating a level of spatial resolution that is unmatched by conventional methods25,26,27.
A large family of genetically-encoded sensors based on green fluorescent protein (GFP) have been developed and characterized to report on a broad variety of physiological markers, including pH, temperature, calcium concentrations, and the ATP/ADP ratio25,28,29,30,31. Included among these are sensors of the glutathione redox potential (EGSH) and hydrogen peroxide (H2O2). While these sensors were developed for applications in redox biology and physiology, they have also been adapted to study xenobiotic-induced oxidative stress. Specifically, the protocol outlined here describes the use of the EGSH sensor roGFP2 and the H2O2 sensor HyPer.
roGFP2 reports on the redox potential of intracellular reduced and oxidized glutathione (GSH/GSSG) through a redox relay involving glutathione peroxidase (GPx), glutaredoxin (Grx), and glutathione reductase (GR) (Figure 1)25,32,33. Glutathione is the predominant cellular antioxidant molecule and is present primarily in its reduced form (GSH) in millimolar concentrations in the cytosol25,34. While EGSH has not been linked to any functional outcome, it is recognized as an important indicator of intracellular oxidative status34. A relatively small increase in the concentration of GSSG results in an increase in EGSH that is detectable by roGFP2. Equally important, monitoring of EGSH using roGFP2 during xenobiotic exposures can potentially reveal much about the mechanism of action at several points in the redox relay and associated pathways, such as the pentose phosphate shunt (Figure 1)35. The second sensor discussed here, HyPer, is an intracellular H2O2 probe derived from the insertion of yellow fluorescent protein (YFP) into the regulatory domain of bacterial H2O2-sensitive transcription factor OxyR136. Although it has previously been considered a damaging reactive oxygen intermediate, H2O2 is increasingly being recognized as an important intracellular signaling molecule under physiological conditions37,38, suggesting that unrecognized roles for H2O2 exist in toxicology as well. For instance, excess H2O2 induced by a xenobiotic exposure could be a precursor to dysregulation in cellular signaling or a shift in bioenergetics.
Both genetically-encoded fluorogenic sensors have been expressed in several established cell lines, including the human epidermoid carcinoma cell line A431 and the human bronchial epithelial cell line BEAS-2B, to observe changes in EGSH and H2O2 in response to a variety of toxicological exposures. These include gaseous pollutants (ozone35), soluble components of particulate matter (1,2 naphthoquinone39,40 and zinc41), and nickel nanoparticles (unpublished data). These studies represent only a small subset of the possible applications of these two sensors. Theoretically, any cell type that is capable of receiving and expressing the DNA of these sensors through conventional molecular biology techniques can be utilized to assess the effects of xenobiotics suspected to alter the cellular oxidative state. To date, one or more of these sensors has been expressed in various prokaryotes and eukaryotes, including several mammalian, plant, bacterial, and yeast cell types25,26,36,42. The readout for both EGSH and H2O2 sensors is a change in the intensity of fluorescence emitted at 510 nm upon excitation with 488 and 404 nm light. This method is widely adaptable across fluorometric platforms, including various types of microscopy (confocal and wide-field) and plate readers. The method presented here allows for sensitive and specific observation of intracellular EGSH and H2O2 in in vitro toxicological systems.

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			<td height="45" style="height:45px;width:216px;">roGFP2 Plasmid</td>
			<td style="width:123px;">University of Oregon</td>
			<td style="width:243px;">N/A</td>
			<td style="width:167px;">Generous gift of S.J. Remington</td>
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			<td height="40" style="height:40px;width:216px;">HyPer Plasmid</td>
			<td style="width:123px;">Evrogen</td>
			<td>FP941</td>
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			<td height="40" style="height:40px;width:216px;">Hydrogen Peroxide</td>
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			<td height="40" style="height:40px;width:216px;">Dithiothreitol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:243px;">10708984001</td>
			<td style="width:167px;"></td>
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			<td height="40" style="height:40px;width:216px;">9,10-Phenanthrenequinone</td>
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			<td height="40" style="height:40px;width:216px;">Black Wall Glass Bottomed Dishes</td>
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			<td height="40" style="height:40px;width:216px;">BEAS-2B cell line</td>
			<td style="width:123px;">American Type Culture Collection</td>
			<td style="width:243px;">CRL-9609</td>
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			<td height="40" style="height:40px;width:216px;">Keratinocyte Basal Medium</td>
			<td style="width:123px;">Lonza</td>
			<td style="width:243px;">192151</td>
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			<td height="40" style="height:40px;width:216px;">Keratinocyte Growth Medium BulletKit</td>
			<td style="width:123px;">Lonza</td>
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			<td height="40" style="height:40px;width:216px;">Excel&#160;</td>
			<td style="width:123px;">Microsoft Office Suite</td>
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			<td height="40" style="height:40px;width:216px;">NIS-Elements AR Imaging Software</td>
			<td style="width:123px;">Nikon</td>
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			<td height="40" style="height:40px;width:216px;">Nikon C1si Confocal Imaging System</td>
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imaging approaches to assessments of toxicological oxidative stress using genetically-encoded fluorogenic sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

The use of roGFP2 and HyPer in detecting changes in EGSH and intracellular H2O2 has been well described previously25,36,42,43 and is demonstrated here. Confocal images of cells expressing roGFP2 at baseline and following addition of H2O2 and DTT are shown in Figure 2. Data from ...

Watch this Scientific Journal Video about Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors at JoVE.com

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

The overall goal of this methodology is to establish the use of genetically-encoded redox sensors for the specific assessment of toxicant-induced perturbations in redox homeostasis. By overcoming the significant limitations of conventional approaches used with oxidative stress, this method will help answer key questions pertaining to toxicological oxidative stress. The main advantage of this technique is that it combines a high degree of specificity and sensitivity with unparalleled spacio-temporal resolution to investigate toxicological oxidative stress in living cells.Perform all imaging analysis using a confocal microscope equipped with laser lines at 404 and 488 nanometers, and environmental controls to maintain an appropriate temperature, humidity, and/or gas concentration suitable for the cells throughout the duration of the experiment. If using high values of relative humidity, use an objective heater and/or heating tape to keep any surfaces that may come in contact with the humidified atmosphere at or slightly above the temperature at which the humidity is generated to prevent condensation. Turn on all microscope components, and set up all equipment required for sequential excitation at 488 and 404 nanometers with emission at 510 nanometers.Ensure all components of the optical configuration are set appropriately for real-time acquisition. Set up a stage-top environmental chamber to maintain constant temperature at 37 degrees celsius, 5%CO2 atmosphere, and greater than 95%humidity. Prior to starting image acquisition, equilibrate the environmental chamber for at least 10 minutes after the initial set-up of all environmental controls.Place the dish of cells on the stage top within the environmental chamber. With the desired objective lens, find the focal plane of cells using the eyepiece and white light and ensure normal morphology. A 1.4 numerical aperture 60 X violet corrected oil-immersed objective lens is commonly used, which permits identification of intracellular compartments while maximizing the optical resolution of the confocal system.Check the fluorescence expression of the cells in the field of view while visualizing the cells under wide-field fluorescence illumination with an appropriate filter set such as flourescian-isothioanate or FITC. At this point, using wide-field illumination while looking through the eyepiece is more convenient because it is easier to move the dish to select a field of cells to study. Once a desired field of view is found, close the environmental chamber.Use the focus maintaining feature to facilitate maintenance of a stable focal plane throughout the study. Next, set up acquisition parameters to ensure optimal assessment of the sensor of interest across the desired exposure period. To do so, adjust the laser power for excitation at 488 nanometers and emission at 510 nanometers.Choose a laser power level that allows visualization of the cells and keep this constant between samples or replicate dishes. Use the confocal controls of the acquisition software to ensure that the selected focal plane has been optimized for maximal florescence emission intensity in the center of the cells by scanning at 488 nanometers while adjusting the Z-plane. Use a high gain setting while adjusting for the Z-plane with the most over-exposed cells.Once the appropriate focal plane has been found, return the gain to a setting that is most optimal for the fluorescence of the reporter being used without over-saturating the pixels being observed. Then, use the gain to fine-tune the baseline fluorescence. With roGFP2, establish the baseline near the upper limit of the instrument without over-saturation, as these cells will lose 510 nanometer fluorescence induced by 488 nanometer excitation when the glutathione redox potential increases.Repeat these steps with excitation at 404 nanometers and emission at 510 nanometers. Gain settings for the 404 nanometer excitation wavelength are opposite to those used with 488 nanometer excitation for each sensor. In general, fluorescence of the 404 nanometer excitation will be considerably lower than that obtainable with the 488 nanometer excitation in cells expressing roGFP2, as a 404 nanometer peek is a relatively minor excitation maximum for this sensor.Set up the acquisition software to sequentially excite the two excitation wavelengths and collect emissions for both at 510 nanometers and at a predetermined time interval throughout the desired length of the experiment. For real-time assessment of experimental parameters, choose at least five to 10 sensor-expressing cells in the field, and establish them as regions of interest to monitor their fluorescence changes during the experiment. Next, dissolve the environmental toxicant, 9, 10-PQ in dimethyl sulfoxide to a concentration of 15 millimolar.Dilute the dissolved toxicant in basal-cell media to a 250 micromolar working solution for a final concentration of 25 micromolar. Additionally, prepare a working solution of hydrogen peroxide for later use in this protocol. Once the experimental parameters have been defined, begin the time course acquisition.Establish a baseline period of at least five minutes prior to starting xenobiotic exposures. Expose the cells to the toxicant being investigated using conventional approaches for in-vitro dosing. Monitor changes in fluorescence during the exposure period.Perform subsequent injections as needed. Take great care not to shift the dish during injections so that the same cells are followed throughout the entire time course while imaged in the same focal plane. At the end of the experiment, expose the cells to the appropriate controls to fully oxidize and reduce roGFP2.The addition of control simuli validates sensor responsiveness at the end of the exposure and confirms that the responses observed are not the direct result of damage to the forport. So, it is especially important in the initial evaluation of a toxin. For these studies, use one millimolar hydrogen peroxide to determine the maximum sensor response.Wait at least five minutes to allow the sensor to respond and then inject a reducing agent such as DTT to reduce the sensor and return it to a level of fluorescence at or near its established baseline. Finally, perform data analysis as described in the text protocol. Shown here are representative results illustrating an established dose response generated using the bronchial epithelial cell line Bees 2B expressing roGFP2.Cursor shown to exposure five micromolar, 25 micromolar, or 100 micromolar doses of hydrogen peroxide. Exposure of bees 2B cells to a previously untested environmental toxicant, 9, 10-PQ induces a gradual but potent increase in the intracellular glutathione redox potential as assessed by roGFP2. Here, each line depicts the raw roGFP2 ratio of individual cells in the dish.Although the test compound produces a maximal response comparable to one millimolar hydrogen peroxide, this known redox cycler caused a change in the sensor that was fully reduced with the addition of five millimolar DTT, suggesting that the sensor remains functional following exposure to this redox-active organic compound. This approach will aid toxicologists in investigating key mechanistic events in toxican-induced oxidative stress, paving the way for more focus studies in the emergent field of redox toxicology across a broad spectrum of toxicants and cell types. After watching this video, you should have a good understanding of how to use genetically encoded sensors and live-cell microscopy to monitor pivotal markers of toxicological oxidative stress with high sensitivity and specificity at unmatched spacio-temporal resolution.

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Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to interference with proper development and may even result in initiation or progression of cancers in certain cases. Because this pathway serves complex and versatile functions, it can be studied extensively through many different approaches. Of these, bioinformatics provides an undeniably cost-efficient, approachable, and user-friendly method of study. Bioinformatics is a useful way to extract smaller pieces of information from large-scale datasets. Through the implementation of various bioinformatics approaches, researchers can quickly, reliably, and efficiently interpret these large datasets, yielding insightful applications and scientific discoveries. Here, a protocol is presented for integration of bioinformatics approaches to investigate the role of Notch signaling in ovarian cancer. Furthermore, bioinformatics findings are validated through experimentation.

Bioinformatics is a useful way to process large-scale datasets. Through the implementation of bioinformatics approaches, researchers can quickly, reliably, and efficiently obtain insightful applications and scientific discoveries. This article demonstrates the utilization of bioinformatics in ovarian cancer research. It also successfully validates bioinformatics findings through experimentation.

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

Modeling the Effects of Hemodynamic Stress on Circulating Tumor Cells using a Syringe and Needle

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed to mechanical stress due to hemodynamic flow. One of these stresses that circulating tumor cells (CTCs) experience is fluid shear stress (FSS). While cancer cells may experience low levels of FSS within the tumor due to interstitial flow, CTCs are exposed, without extracellular matrix attachment, to much greater levels of FSS. Physiologically, FSS ranges over 3-4 orders of magnitude, with low levels present in lymphatics (&#60;1 dyne/cm2) and the highest levels present briefly as cells pass through the heart and around heart valves (&#62;500 dynes/cm2). There are a few in vitro models designed to model different ranges of physiological shear stress over various time frames. This paper describes a model to investigate the consequences of brief (millisecond) pulses of high-level FSS on cancer cell biology using a simple syringe and needle system.

Here we demonstrate a method to apply fluid shear stress to cancer cells in suspension to model the effects of hemodynamic stress on circulating tumor cells.

During metastasis, cancer cells from solid tissues, including epithelia, gain access to the lymphatic and hematogenous circulation where they are exposed ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.