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 authors have nothing to disclose.

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

9.7K visualizações.  Boston University School of Medicine. 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 i...