Name: long-term live-cell imaging to assess cell fate in response to paclitaxel
Uploaded: 2018-05-14T13:00:00
Description: Watch this Scientific Journal Video about Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel at JoVE.com

AFB, MAV and NJG would like to thank Ryan Quinton for comments on the manuscript and Adrian Salic for the TDRFP-NLS construct. AFB and MAV are funded by the NIGMS Biomolecular Pharmacology Training Grant 5T32GM008541. NJG is a member of the Shamim and Ashraf Dahod Breast Cancer Research Laboratories and is supported by NIH grants GM117150 and CA-154531, the Karin Grunebaum Foundation, the Smith Family Awards Program, the Searle Scholars Program, and the Melanoma Research Alliance.

This protocol focuses on using the non-transformed and chromosomally stable retinal pigmented epithelial (RPE-1) cell line for live-cell imaging experiments. However, this protocol can be adapted to any adherent cell line so long as cell culture conditions are adjusted as necessary. All procedures must adhere to institutional biosafety and ethical guidelines and regulations.
1. Preparing Cells for Live-cell Imaging
<ol>
	<li>Use freshly thawed and early passage RPE-1 cells expressing...

The most critical aspect of long-term live-cell imaging is ensuring the health of the cells being imaged. It is essential that cells be exposed to minimal extraneous environmental stressors, such as substandard conditions regarding temperature, humidity, and/or CO2 levels. It is also important to limit photodamage from fluorescent excitation illumination, as imaging stress is known to affect cell behavior50. Limiting photodamage can be achieved in multiple ways as detailed elsewhere<sup...

Anti-mitotic drugs have long been used in the chemotherapeutic regimens of various types of solid tumors and often show great efficacy1,2,3. Mechanistically, these drugs disrupt normal mitotic progression and promote mitotic arrest in rapidly proliferating cancer cells. However, cell fate in response to mitotic arrest is highly variable: while a fraction of cells undergoes cell death directly from mitosis, others exit out of mitosis and return to interphase as tetraploid cells (a process termed mitotic slippage)4,5,6,7,8. These interphase cells can execute apoptosis, undergo permanent cell cycle arrest, or even re-enter the cell cycle4,5,6,7,8,9,10,11. Cells that evade mitotic cell death by slipping into interphase, only to re-enter the cell cycle following drug removal, may therefore contribute to the re-emergence of cancer cell populations. Moreover, cells that slip from mitosis are tetraploid, and tetraploidy is known to promote chromosome instability that drives tumor relapse12,13,14,15. Defining the factors that control cell fate in response to anti-mitotic drug treatments is therefore critical to optimize current therapeutics.
In this protocol, we describe methods to directly observe and study the fate of cells that undergo prolonged mitotic arrest in response to the anti-mitotic drug paclitaxel. Paclitaxel is an established therapeutic in the clinic and has proven highly efficacious in many tumors types, including those of the breast, ovaries, and lungs16,17,18,19,20. Paclitaxel, which is a plant alkaloid derived from the bark of the Yew tree, stabilizes microtubules and thus prevents their dynamicity21,22. While dampening of microtubule dynamics by paclitaxel does not affect cell cycle progression from G1 through G2, the drug does lead to sustained activation of the spindle assembly checkpoint during mitosis by hindering kinetochore-microtubule attachment (reviewed in depth here23,24)25. As a consequence, anaphase onset is prevented in paclitaxel-treated cells and results in a prolonged mitotic arrest.
This protocol will first describe approaches to identify mitotic cells in live-cell imaging experiments. Mitosis can be visualized in adherent tissue culture cells due to two noticeable cell biological changes. First, chromosomes become highly condensed immediately prior to nuclear envelope breakdown. While often detectable by standard phase-contrast microscopy, chromosome condensation can be more clearly detected using fluorescent tags that label chromosomes (e.g. fluorescently-labeled histone proteins). Second, mitotic cells can also be identified by the dramatic morphological changes that result from cell rounding.
This protocol will then demonstrate how to use live-cell imaging approaches to track the fates of cells experiencing prolonged mitotic arrest. Cells arrested in mitosis undergo one of three distinct fates. First, cells can undergo cell death during mitosis. This phenomenon is readily visualized by light microscopy, as dying cells are observed to shrink, bleb, and/or rupture. Second, cells can exit from mitosis and return back to interphase without chromosome segregation or cytokinesis, a process termed mitotic slippage. The decondensation of chromosomes and/or the flattening of the mitotic cell readily identifies this process. Cells that slip from mitosis also often display irregular, multi-lobed nuclei and frequently harbor several micronuclei5. Third, cells arrested in mitosis can initiate anaphase and proceed through mitosis after a long delay. While uncommon at higher drug concentrations, this behavior suggests that the arrested cells may have satisfied the spindle assembly checkpoint, or that the spindle assembly checkpoint is partially weakened or defective. Anaphase onset can be visualized by chromosome segregation and subsequent cytokinesis using live-cell imaging.
Live-cell imaging methods to track the fate of cells that evade mitotic cell death by undergoing mitotic slippage will also be described. Cells that undergo mitotic slippage either die in the subsequent interphase, trigger a durable G1 cell cycle arrest, or re-enter the cell cycle to initiate a new round of cell division4. An approach using the FUCCI (fluorescent ubiquitination-based cell cycle indicator) system to determine the fraction of cells that re-enter the cell cycle following mitotic slippage will be described. FUCCI allows for the direct visualization of the G1/S transition and can be used in conjunction with long-term live-cell imaging both in vitro and in vivo26,27. The FUCCI system takes advantage of two fluorescently labeled proteins, truncated forms of hCdt1 (chromatin licensing and DNA replication factor 1) and hGeminin, whose levels oscillate based on cell cycle position. hCdt1 (fused to a red fluorescent protein) is present at high levels during G1 phase where it acts to license DNA for replication, but is ubiquitinated by the E3 ubiquitin ligase SCFSkp2 and degraded during S/G2/M phases to prevent re-replication of DNA26. By contrast, hGeminin (fused to a green fluorescent protein), is an inhibitor of hCdt1 whose levels peak during S/G2/M, but is ubiquitinated by the E3 ubiquitin ligase APCCdh1 and degraded at the end of mitosis and throughout G126. Consequently, FUCCI delivers a straightforward fluorescence readout of cell cycle phase, as cells exhibit red fluorescence during G1, and green fluorescence during S/G2/M. The FUCCI system is a significant advance over other approaches (such as bromodeoxyuridine staining) to identify proliferative cells, because it does not require cell fixation and allows for single cell imaging without the need for additional pharmacological treatments to synchronize cell populations. Though not discussed in this protocol, additional live-cell sensors have also been developed to visualize cell cycle progression, including a helicase B sensor for G128, DNA ligase-RFP29 and PCDNA-GFP30 sensors for S-phase, and the recent FUCCI-4 sensor, which detects all stages of the cell cycle31.
Finally, a live-cell imaging method to detect nuclear envelope rupture will be described. Recent studies have revealed that the nuclear envelopes of cancer cells are unstable and prone to bursting, thereby allowing the contents of the nucleoplasm and cytoplasm to intermix. This phenomenon, termed nuclear rupture, can promote DNA damage and stimulation of the innate immune response32,33,34,35,36,37,38,39,40,41,42,43,44. While the underlying causes of nuclear rupture remain incompletely characterized, it is known that deformations in nuclear structure correlate with an increased incidence of nuclear rupture42. One well-known effect of paclitaxel treatment is the generation of strikingly abnormal nuclear structures following mitosis; as such, a method using live-cell imaging to quantify nuclear rupture will be described, while also exploring if paclitaxel treatment increases the frequency of nuclear rupture events. Nuclear rupture can be detected by the observed leakage of a nuclear-targeted fluorescent protein into the cytoplasm (e.g. a tandem dimer repeat of RFP fused to a nuclear localization signal, TDRFP-NLS). This leakage is distinctly visible by eye, which enables simple quantitation of rupture events.
This protocol requires a widefield epifluorescence microscope that is equipped with an encoded stage and autofocusing software. The encoded stage allows for precise automated movement to defined X-Y coordinates, while autofocus software maintains cells in focus for the duration of the imaging period. In addition, this protocol requires equipment to maintain cells at 37 &#176;C with humidified 5% CO2 atmosphere. This can be achieved by enclosing the entire microscope within a temperature and atmosphere controlled enclosure, or by using stage-top devices that locally maintains temperature and environment. The phase-contrast objective used in this protocol is a plan fluor 10x with a numerical aperture of 0.30. However, 20X objectives are also sufficient to identify both rounded mitotic and flattened interphase cells in a single focal plane. If performing phase-contrast imaging (as described in this method), the cover can be either glass or plastic. If differential interference contrast (DIC) microscopy is used, it is imperative to use a glass cover to prevent depolarization of light.

<table>
	<tbody>
		<tr>
			<td>phenol red-free, DMEM:F12 media</td>
			<td>Hyclone</td>
			<td>SH3027202</td>
			<td></td>
		</tr>
		<tr>
			<td>10% fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10438026</td>
			<td></td>
		</tr>
		<tr>
			<td>100 IU/ml penicillin, and 100 mg/ml streptomycin.</td>
			<td>Gibco</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gemini</td>
			<td>50-753-3104</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well glass bottomed imaging dish</td>
			<td>Cellvis</td>
			<td>P12-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Paclitaxel</td>
			<td>TSZ Chem</td>
			<td>RS036</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>NC0215085</td>
			<td></td>
		</tr>
		<tr>
			<td>Environmental Chamber</td>
			<td>In Vivo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5% CO2</td>
			<td>Airgas</td>
			<td>Z03NI7432000201</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tubes</td>
			<td>Fisherbrand</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm polystyrene tissue culture plates</td>
			<td>Falcon</td>
			<td>08772E</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Disposable Serological Pipette</td>
			<td>Fisherbrand</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>5804R 15 amp Centrifuge</td>
			<td>Eppendorf</td>
			<td>97007-370</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 microliters Pipette</td>
			<td>Gilson</td>
			<td>Pipetman Classic</td>
			<td></td>
		</tr>
		<tr>
			<td>20 microliters Pipette</td>
			<td>Gilson</td>
			<td>Pipetman Classic</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 Pipette Tips</td>
			<td>Sharp</td>
			<td>p1126</td>
			<td></td>
		</tr>
		<tr>
			<td>p20 Pipette Tips</td>
			<td>Sharp</td>
			<td>P1121</td>
			<td></td>
		</tr>
		<tr>
			<td>RPE-1 Cells</td>
			<td>ATCC</td>
			<td>CRL-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Galaxy 170S</td>
			<td>Eppendorf</td>
			<td>CO17011005</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Boy</td>
			<td>Integra</td>
			<td>156401</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>0267110</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Ti-E-PFS Inverted Microscope for Live Cell Imaging</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MEA53100</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior X-Y Stage System and White Light LED Illuminator</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>500-H117P1N4</td>
			<td></td>
		</tr>
		<tr>
			<td>Prior Proscan 3 Controller XYZ with Encoders</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>500-H31XYZE</td>
			<td></td>
		</tr>
		<tr>
			<td>Andor Digital Camera</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>D10NLC</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon NIS Elements AR Software</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MQS31000</td>
			<td></td>
		</tr>
		<tr>
			<td>SOLA LED Illuminator for Fluorescence</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>SOLA-E</td>
			<td></td>
		</tr>
		<tr>
			<td>InVivo Environmental Chamber with CO2 Flow Control</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>Ti-IVS-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-ND6-PFS Perfect Focus Motorized Nosepiece</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MEP59391</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-C System Condenser Turret</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MEL51000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-C LWD LWD Lens Unit for System Condenser Turret</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MEL56200</td>
			<td></td>
		</tr>
		<tr>
			<td>Te-C LWD Ph1 Module</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MEH41100</td>
			<td></td>
		</tr>
		<tr>
			<td>CFI Plan Fluor DLL 10X Objectivena 0.3 wd 16mm</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MRH10101</td>
			<td></td>
		</tr>
		<tr>
			<td>CFI Super Plan Fluor ELWD 20xc ADM Objective</td>
			<td>Micro Video Instruments, Inc.</td>
			<td>MRH48230</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Using the protocol described above, RPE-1 cells expressing H2B-GFP were treated with an anti-mitotic or vehicle control (DMSO) and analyzed by live-cell imaging. Analysis revealed that 100% of control mitotic RPE-1 cells initiated anaphase an average of 22 min after entering mitosis (Figure 1A, 1D). By contrast, RPE-1 cells treated with paclitaxel exhibited a profound mitotic arrest lasting several hours (Figure 1D</stron...

Watch this Scientific Journal Video about Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel at JoVE.com

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

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 ...

The overall goal of this procedure is to use live-cell imaging to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. This method enables us to visualize how individual cells respond to different doses of the anti-mitotic drug paclitaxel. The main advantage of this technique is that it provides a wealth of information on single cells or whole populations that is unobtainable from fixed-cell imaging or population-based assays.Demonstrating this method is Marc Vittoria, a MD/PhD candidate in the Ganem Laboratory. To begin, clean the glass bottom of the imaging dish with optical cleaner to remove any fingerprints or dust that may interfere with imaging. Use sufficient optical cleaner to wet the entire glass surface.Place the glass-bottom dish with cells on the microscope stage in the imaging dish adaptor. Remove the plastic cover from the dish, and cover the dish with a glass-topped chamber. Ensure the chamber has a valve to allow a steady flow of air consisting of 5%carbon dioxide.To humidify the 5%carbon dioxide, the gas is flowed through tubing inserted into a sterile water bath housing. This allows for the gas to become equilibrated to 95%humidity. Initiate and calibrate the encoded stage using the software program controlling the microscope.This will ensure accurate XY coordinates and prevent focal drift. Next, perform the Kohler illumination by raising the condenser and closing the aperture so that the field is dark. Lower the condenser until a crisp hexagonal shape of light is visible.Then, use the centering screws to center the hexagon of light in the field of view. Once the hexagonal shape is visible, open the aperture until the whole field of view is illuminated. Engage the microscope's auto-focus to ensure that all points are maintained in focus through the duration of the experiment.Use the acquisition software to select several non-overlapping fields of view from each well for imaging. Select imaging regions where cells have adhered well to the glass bottom and are between 50 and 70%confluent. Avoid areas of clumped cells, as this will make subsequent analysis difficult.To assess mitotic cell fate, set the imaging software to collect images from each selected field of view every 10 minutes for up to 96 hours. Unperturbed mitosis lasts 20 to 40 minutes, and thus 10-minute intervals will provide enough sampling to identify when cells divide. To identify nuclear rupture, which is a transient event, acquire images every five minutes.Identify cells that enter mitosis by observation of cell rounding, using phase-contrast optics, and/or chromosome condensation, using human histone H2B fused to GFP. Both cell rounding and chromosome condensation are readily visualized by eye. Annotate the time when the cell enters mitosis.Continue tracking the cell until it reaches its fate. Control cells should efficiently align their chromosomes and enter anaphase within one hour. Visualize anaphase by phase-contrast optics as the cell begins to pinch into two or through visualization of poleward-moving chromosomes labeled with H2B-GFP.Annotate the time when the cell undergoes anaphase. By contrast, cells treated with paclitaxel will remain rounded with condensed chromosomes for several hours. Identify mitotically arrested cells that undergo cell death.Cells that die during mitosis are visualized by phase-contrast microscopy, as cells will bleb, shrink, and/or rupture. If imaging H2B-GFP, the chromosomes will also fragment during cell death. Identify mitotically arrested cells that undergo cell slippage.Cells that undergo mitotic slippage are observed by phase-contrast microscopy, as they flatten back out into interphase and decondense chromosomes without undergoing anaphase. These cells give rise to large tetraploid cells that are often multinucleated. Continue tracking cells from the original field of view.Once a whole field of view is tracked, move to a separate field of view acquired from the same well and continue tracking the cells. Assess cell fate following mitotic slippage using the non-transformed and chromosomally stable retinal pigmented epithelial cell line expressing the FUCCI system. To confirm that the FUCCI system is working properly, validate that control cells alternate expression of the red and green fluorescent proteins appropriately from analysis of the live-cell imaging data.Control cells should transition from exhibiting entirely nuclear red fluorescence to exhibiting entirely nuclear green fluorescence during interphase as cells progress from G1 to S phase. Cells should continue exhibiting green fluorescence throughout the completion of mitosis. Immediately following mitosis, cells should once again exhibit entirely red fluorescence during interphase.Identify cells arrested in mitosis using phase-contrast microscopy, and track them until they undergo mitotic slippage. Cells that slip out of mitosis and back into interphase will change from exhibiting green fluorescence during mitosis to red fluorescence during G1 phase. Track these slipped cells using phase-contrast and epifluorescent imaging by eye to assess their cell fate.Identify cells that re-enter the cell cycle. These cells are identified by the red-to-green change in fluorescence expression using the FUCCI system that indicates the G1/S transition. Identify cells that undergo G1 cell cycle arrest.These cells are identified by expression of red fluorescence that persists for greater than 24 hours. Also, identify cells that die in interphase. These cells are identified by cell rupture, blebbing, and shrinking using phase-contrast imaging.Image the cells as before. The fluorescence signal from the tandem dimer repeat of RFP fused to a nuclear localization signal, or TDRFP-NLS, and H2B-GFP should co-localize during interphase. Mitosis is visualized by cell rounding using phase optics and/or chromosome condensation by H2B-GFP.Upon mitosis, the nuclear envelope will break down and the TDRFP-NLS signal will become cytoplasmic. Following mitosis, the nuclear envelope will reform in the daughter cells and the TDRFP-NLS signal will become nuclear. Track daughter cells throughout the subsequent interphase by live-cell imaging.Identify nuclear rupture events by observing a transient burst of nuclear-localized TDRFP-NLS into the surrounding cytoplasm. Within minutes, the nuclear envelope will be repaired and the TDRFP-NLS will be relocalized to the nucleus. Shown here are representative images of a slipped cell turning green and entering S phase after slipping from paclitaxel treatment.A majority of cells that slipped from paclitaxel treatment underwent a G1/G0 arrest and stayed red for more than 48 hours without undergoing apoptosis. Cells that are red post-slippage will also undergo apoptosis, as represented by this image. Nuclear envelope rupture increases in paclitaxel-treated cells.TDRFP-NLS and H2B-GFP cells treated with 10-nanomolar paclitaxel have an increased nuclear rupture in which RFP will be delocalized from the nucleus to the cytoplasm in interphase cells. After watching this video, you should have a solid understanding of how to perform long-term live-cell imaging to assess cell fate in response to various drug treatments.

long-term-live-cell-imaging-to-assess-cell-fate-response-to

Research

JoVE Journal

Cancer Research

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-&#946; is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-&#946;/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-&#946; displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-&#946; signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-&#946; signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Live Cell Imaging of the TGF-	&amp;beta;/Smad3 Signaling Pathway &lt;em&gt;In Vitro&lt;/em&gt; and &lt;em&gt;In Vivo&lt;/em&gt; Using an Adenovirus Reporter System

Here, we present a protocol for live cell imaging of TGF-&#946;/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Establishing Cell Lines Overexpressing DR3 to Assess the Apoptotic Response to Anti-mitotic Therapeutics

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell lines or increasing its expression with overexpression cell lines. Transient and stable transfection are two methods that are often used for exogenous gene expression. Transient transfection is only useful for short-term expression, whereas stable transfection allows exogenous genes to be integrated into the host cell genome where it will be continuously expressed. As a result, stable transfection is usually employed for research into long-term genetic regulation. Here we describe a simple protocol to generate a stable cell line overexpressing tagged death receptor 3 (DR3) to explore DR3 function. We picked single clones after a retroviral infection in order to maintain the homogeneity and purity of the stable cell lines. The stable cell lines generated using this protocol render DR3-deficient HT29 cells sensitive to antimitotic drugs, thus reconstituting the apoptotic response in HT29 cells. Moreover, the FLAG tag on DR3 compensates for the unavailability of good DR3 antibody and facilitates the biochemical study of the molecular mechanism by which antimitotic agents induce apoptosis.

Establishing a stable cell line overexpressing a gene of interest to study gene function can be done by stable transfection-picking single clones after transfecting them via retroviral infection. Here we show that HT29-DR3 cell lines generated in this way elucidate the mechanisms by which death receptor 3 (DR3) contributes to antimitotics-induced apoptosis.

Studying the function of a gene of interest can be achieved by manipulating its level of expression, such as decreasing its expression with knockdown cell ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Quantifying Telomere Length Using Non-Radioactive Chemiluminescence Detection

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening occurs due to a problem with end replication and the absence of the telomerase enzyme, which is responsible for maintaining telomere length. Interestingly, telomeres also shorten in response to various internal physiological processes, like oxidative stress and inflammation, which may be impacted due to extracellular agents like pollutants, infectious agents, nutrients, or radiation. Thus, telomere length serves as an excellent biomarker of aging and various physiological health parameters. The TAGGG telomere length assay kit is used to quantify average telomere lengths using the telomere restriction fragment (TRF) assay and is highly reproducible. However, it is an expensive method, and because of this, it is not employed routinely for large sample numbers. Here, we describe a detailed protocol for an optimized and cost-effective measurement of telomere length using Southern blots or TRF analysis and non-radioactive chemiluminescence-based detection.

Author Spotlight: Optimization of Performance Parameters of the TAGGG Telomere Length Assay  

Here, we describe in detail the protocol for quantifying telomere length using non-radioactive chemiluminescent detection, with a focus on the optimization of various performance parameters of the TAGGG telomere length assay kit, such as buffer quantities and probe concentrations.

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...

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Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell lines. PDO models can be generated by culturing patient tumor cells in extracellular basement membrane extracts (BME) and plating them as three-dimensional domes. However, commercially available reagents that have been optimized for phenotypic assays in monolayer cultures often are not compatible with BME. Herein, we describe a method to plate PDO models and assess drug effects using an automated live-cell imaging system. In addition, we apply fluorescent dyes that are compatible with kinetic measurements to quantify cell health and apoptosis simultaneously. Image capture can be customized to occur at regular time intervals over several days. Users can analyze drug effects in individual Z-plane images or a Z Projection of serial images from multiple focal planes. Using masking, specific parameters of interest are calculated, such as PDO number, area, and fluorescence intensity. We provide proof-of-concept data demonstrating the effect of cytotoxic agents on cell health, apoptosis, and viability. This automated kinetic imaging platform can be expanded to other phenotypic readouts to understand diverse therapeutic effects in PDO models of cancer.

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived tumor organoids are a sophisticated model system for basic and translational research. This methods article details the use of multiplexed fluorescent live-cell imaging for simultaneous kinetic assessment of different organoid phenotypes.

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...