We thank Joshua Marcus for technical support and Jolien Tyler, Ph.D., Director of the Richard J. McIntosh Light Microscopy Core Facility, for technical advice. This work was supported by funds from the University of Colorado Boulder and the University of Colorado Boulder Graduate School to J.D.O. R.T.B. is partially supported by pre-doctoral training grant from the NIH (T32 GM008759). We thank Karyopharm Therapeutics, Inc. for selinexor and Merck Serono for Kinesin-5 inhibitor. FUCCI plasmids are from Atsushi Miyawaki (RIKEN, Japan) via MTA. mCherry-BP1-2 was from Addgene. HeLa expressing H2b-mCherry and &#946;-tubulin-EGFP are from Daniel Gerlich (IMBA, Austrian Academy of Sciences, Austria).

&#160;The following protocol uses parameters defined by the experiments in Figures 4 and 6 regarding acquisition settings and experimental conditions. Many of these parameters can be modified to fit other experiments (i.e., exposure times, binning, fluorescent channels, etc.). All procedures must adhere to institutional guidelines and regulations and be approved by the institutional biosafety committee. Microscope manufacturer websites contain excellent information for live cell imaging.
1. Microscopes and Imaging Software
<ol>
	<li>Perform live cell imaging on a wide variety of inverted microscopes. The most common microscopes are widefield epifluorescence and spinning-disc confocal. Here, use an automated and motorized widefield epifluorescence microscope with 200 watt metal halide light source.</li>
	<li>Obtain a stage-top or microscope environmental chamber. Here, use a stage-top environmental chamber.</li>
	<li>Use commercial software to operate microscopes with utilities to execute time-lapse microscopy.</li>
	<li>Use commercially available software or ImageJ for image analysis. Many other analysis tools are available commercially, and there are custom-made programs many of which have been published8,18.</li>
</ol>
2. Visualizing Cellular Processes and Phenotypic Responses
<ol>
	<li>Visualize cells with brightfield microscopy. Differential interference contrast and phase contrast alone can be very informative to study responses to anti-cancer drug responses. These processes can include mitosis, cell motility and apoptosis.</li>
	<li>Visualize cell structures, organelles and processes with fluorescent biosensors. Fluorescent probes are informative in tracking specific subcellular processes. These can include microtubule dynamics, mitochondrial and endoplasmic reticulum dynamics, protein accumulation and localization, and molecular signaling (e.g., phosphorylation, calcium).</li>
</ol>
3. Preparation of Samples
<ol>
	<li>Grow cells in cell culture certified dishes in a standard, humidified cell culture incubator (e.g., 37 oC, 5% CO2, 80% relative humidity).
	<ol>
		<li>Grow HT1080 cells in MEM with EBSS. Supplement the media with 10% v/v FBS, 1% v/v Pen/Strep, 1% v/v sodium pyruvate and 1% v/v non-essential amino acids.</li>
	</ol>
	</li>
	<li>Two days prior to imaging, plate 50,000 HT1080 FUCCI cells into 3 wells of a 12-well #1.5 glass bottom dish in a certified sterile laminar-flow hood. Adjust the number of cells plated to achieve ~60% confluence for the start of the experiment. Depending on the cell line and nature of experiment the cell density may be less. 
	Note: Cell density may have profound influence on the growth of the cell line and on the experimental results. Count cells to minimize experiment-to-experiment variability due to density.
	<ol>
		<li>Depending on the environmental chamber and the necessary conditions for the experiment, plate cells in single well dishes (typically 35- or 60-mm), 4 well 35-mm dishes, 6-, 12- or 24-well cell culture plates, or coverslip slides in various formats. Use glass-bottom plates with #1.5 glass as most objective lenses are optimized for this thickness of glass, and imaging through cell culture plastic is very poor. 
		Note: Some cell lines do not grow and survive on glass. In these cases, the glass can be coated in order to enhance cell adherence (e.g., poly-lysine or collagen). Some companies manufacture optical cell culture plastic, it must be determined empirically if this is a viable option.</li>
	</ol>
	</li>
</ol>
4. Environmental Chamber Set-up
<ol>
	<li>Prior to any experimentation, set-up the environmental chamber so that it operates at ~80% humidity and so the temperature at the sample position is 37 oC. Most cultured cells grow in 5% CO2/balance air atmosphere due to sodium bicarbonate buffer in the medium. Depending on the set-up, either set the environmental controller to 5% CO2 and it will mix 100% CO2 with air, or use pre-mixed, certified 5% CO2/balance air gas. Follow manufacturer directions for gas flow-rates. 
	Note: Some cell lines grow in CO2-independent medium in which case they can be maintained without CO2. Specifically designed imaging media is also available that limits the addition of autofluorescent compounds. Growth in theses mediums for the desired cell type must be determined empirically beforehand.</li>
	<li>Prior to imaging, ensure the water reservoir is filled (following manufacturer&#39;s directions) with sterile distilled water. Turn on the environmental chamber to the desired temperature setting and place it into the microscope stage inset. To save gas, do not start the flow at this point. 
	Note: If there is any free space between the stage-top chamber and stage inset and/or any tension on connection cords to the chamber it may introduce motion artifacts into the experiment.
	<ol>
		<li>Lay pieces of paraffin film over the edges of the stage inset opening prior to inserting the chamber into it to couple the chamber to the stage, and make sure that no connections are pulling on the chamber.</li>
	</ol>
	</li>
	<li>Allow the chamber to equilibrate to 37 oC. Maintain a stable temperature to prevent temperature fluctuations during imaging which can affect cell physiology and introduce drift. Reaching temperature equilibration typically requires 30 min&#160;to 1 hr, depending on the environment.
	<ol>
		<li>Insert a &#39;dummy&#39; dish with water in the wells into the chamber while warming. An imaging dish filled with water mimics the sample, helping ensure the chamber is sufficiently warmed and stabilized. Filling the chamber with pre-warmed sterile water reduces the time required to temperature stabilization and minimizes the temperature decrease upon addition of the experimental sample.</li>
	</ol>
	</li>
</ol>
5. Microscope Set-up
<ol>
	<li>Turn on the microscope, associated computer, and any required peripherals.
	<ol>
		<li>Depending on the light source, wait to turn it on until needed (e.g., LED).</li>
	</ol>
	</li>
	<li>With the objective turret in a low position, select the 20X 0.7 NA objective to be used.</li>
	<li>Position the sample over the objective - this will make it easier to find the cells when the sample is in the chamber.</li>
	<li>Define imaging parameters at this time if they are known from previous experiments.</li>
</ol>
6. Transporting Cells to Microscope and into the Chamber
<ol>
	<li>Transport the cells to be imaged from the incubator to the environmental chamber. Ensure that this is done quickly to limit the effects of a comparatively low CO2 atmosphere and decreased temperature on the cells. Follow all institutional biosafety guidelines for sample transport and clean-up in case of a spill.
	<ol>
		<li>Place the cells in a sealed Styrofoam container or insulated bag to avoid large environmental changes.</li>
	</ol>
	</li>
	<li>Insert the sample imaging dish into the chamber following manufacturer&#39;s directions.</li>
	<li>After the cells have been secured within the environmental chamber, seal the chamber to maintain a stable environment and immediately turn on the source(s) of atmospheric gas.
	<ol>
		<li>As some environmental chambers do not utilize a tight, sealed lid, to retard evaporation, layer sterile, embryo-certified mineral oil on top of the growth medium. Wrap gas-permeant paraffin film around the perimeter-edges of the sample dish being careful not to obstruct the glass imaging area. Localized, secondary humidification methods can be employed. Condensation on the lid of the sample dish can diminish performance of brightfield microscopy.</li>
	</ol>
	</li>
	<li>In order to minimize the effects of thermal drift early during the experiment, wait 30 min&#160;before starting the time-lapse. The required time varies based on imaging dish size and other factors. Determine empirically.</li>
</ol>
7. Setting up the Imaging
<ol>
	<li>Select imaging conditions that will best represent the data. Take precautions to avoid phototoxicity by limiting exposure times, using lower intensity light and selecting probes that are excited by longer wavelengths of light.</li>
	<li>Define x, y and z-plane and desired wavelength(s) for each position to be imaged. The time resolution is limited by the number of positions and wavelengths. For long-term time-lapse of most cellular processes, acquire one image every 10 - 20 min; higher time resolution (short intervals, e.g.,1 min) provides more data points and therefore more robust cell tracking, but also results in more integrated light exposure and larger data sets.
	<ol>
		<li>As HT1080 FUCCI have dynamic green and red fluorescent proteins (see Representative Results), use the following exposure times: FITC/GFP - 50 msec, Texas Red/TRITC - 40 msec, Brightfield - 20 msec. Use a 2 x 2 bin.</li>
	</ol>
	</li>
	<li>Enable software controlled autofocusing using the default parameters under advanced settings. Define an autofocus range of 10 &#181;m with the recommended step size. Be sure to do this before starting the time-lapse. Autofocus with brightfield images and never with fluorescence to reduce phototoxicity and photobleaching.
	<ol>
		<li>Use software controlled autofocusing systems on any microscope with a motorized stage, and directly focus on the sample. However, they can limit the acquisition speed of the experiment. Hardware controlled continuous focusing systems work well for time-lapse microscopy and improve speed, allowing for more positions to be imaged or a higher rate of acquisition. However, they rely on detection of the air-glass interface and may lose focus of the sample with variations in glass thickness across the well.</li>
	</ol>
	</li>
	<li>Replace half of the media with media containing the desired drug concentration that has been warmed to 37 oC. Partial media replacement helps to reduce thermal drift. The conditions in this experiment are Vehicle (DMSO), 1 &#181;M selinexor and 10 &#181;M PD0332991. 
	Note: This step can also be done after starting the acquisition by pausing and restarting the experiment. This allows for longitudinal tracking of pre- and post-drug responses of single cells. If drug stability or metabolism is a concern, pausing and replacing the media can be done with the same method. To test for drug degradation or metabolism, media from treated cells can also be placed on na&#239;ve cells to test drug action.</li>
	<li>Start the time-lapse.</li>
	<li>As the time-lapse runs, ensure that the imaged fields remain in focus and the chamber maintains a humid 37 oC.
	<ol>
		<li>Adjust focus as needed by pausing the acquisition during the time gap between time points.</li>
	</ol>
	</li>
	<li>If necessary, add water to the chamber during longer experiments. Avoid cooling the chamber by adding pre-warmed, sterile distilled water at 37 oC.</li>
</ol>
8. Ending the Time-lapse
<ol>
	<li>When the experiment has run to completion, stop the acquisition if it was not set to stop automatically.</li>
	<li>Ensure that the time-lapse has been saved properly onto the hard drive, although most software packages automatically save during the acquisition if not finished properly data can be lost.</li>
	<li>Remove the chamber from the microscope and dispose of the cells into biohazardous waste following approved institutional biosafety procedures.</li>
</ol>
9. Longitudinal Tracking and Analysis of Time-lapse Data
<ol>
	<li>Choose the methodology for analysis that is appropriate for the biological processes of interest. Many plugins for ImageJ, programs using MatLab, and custom platforms have been produced for specific applications. The following methodology covers tracking of nuclei and analysis of nuclear fluorescent probes as demonstrated in Figures 4 and 5.</li>
	<li>Open the .tif image stacks for the utilized channels in ImageJ. Alternatively, open native files from the acquisition program directly in ImageJ using the Bioformats plugin.</li>
	<li>Draw a region of interest (ROI) within the nucleus of one cell using the brightfield image or the nuclear label channel (if used) and add it to the ROI manager. Note: If the imaged cells have a nuclear label (e.g., histone H2B-EGFP), then automatic cell tracking may be utilized to create regions of interest (ROIs) representing individual nuclei through time.</li>
	<li>Proceed to the next time point and position the ROI within the same nucleus. Add the ROI to the ROI manager.</li>
	<li>Continue to track and make ROIs for the individual cell until there is a cellular event (mitosis, apoptosis, etc.) or the cell can no longer be tracked (i.e., moves out of frame or the experiment ends).</li>
	<li>When ROIs have been established for cells through the time they are in the field, superimpose them onto the fluorescent channels of interest. Measure the mean intensity of the fluorescence in each channel for every time point. Save the ROI list for later use.
	<ol>
		<li>Various measurements can be made within the ROI for applications in different experiments. Click Analysis &#8594; Set Measurements&#8230; to bring up a menu to choose the analytical method (e.g., mean intensity within the ROI, integrated intensity, etc.).</li>
	</ol>
	</li>
	<li>Note cell fates for individual cells (e.g., apoptosis, division, survival) while tracking. This allows for creation of survival curves and further population analysis.</li>
	<li>With the mean intensity for both channels, create a scatter plot to display cell cycle dynamics in response to therapeutics (Figure 5B, C). 
	Note: Plots can be created for individual cells over time or averaged over a population of tracked cells. Quantitative imaging can be critical for establishing complex responses and cell relationships in response to cancer therapeutics. Long-term time-lapse microscopy, longitudinal tracking and data analysis is a multi-step process, with many options for the type of microscopy and analysis tools, and follows the general outline provided in Figure 1.</li>
</ol>

The authors have no conflicts of interest to disclose.

Advantages of Time-lapse Microscopy and Longitudinal Tracking
The microscope is an ideal instrument for longitudinal studies of drug response as it allows investigators to track individual cells and their fates as well as the entire population. Variability in drug response within a population of cells is a major issue for anti-cancer therapeutic design. Longitudinal tracking of single cells allows investigators to observe this variability and begin to understand the underlying...

Live-cell microscopy and longitudinal tracking of single cells is not a new technique. From the earliest microscopes, enthusiasts and scientists have observed and studied single cells and organisms, their behaviors, and development1-3. A famous example from the late David Rogers at Vanderbilt University in the 1950s shows a human neutrophil in a blood smear chasing a Staphylococcus aureus bacterium and eventually the process of phagocytosis4. This live-cell movie is an excellent illustration of how multiple processes can be observed and correlated in a single experiment: sensing of a chemical gradient, mechanics and speed of cell motility, cell shapes dynamics, adhesion, and phagocytosis of a pathogen.
The advent of fully automated microscopes and highly sensitive digital cameras has resulted in an increasing numbers of investigators using microscopy to ask fundamental questions in cell biology ranging from how cells move5,6 and divide7,8 to organelle dynamics and membrane trafficking9-11. Non-fluorescent, brightfield microscopy, including phase-contrast (PC), which garnered the Nobel Prize for Frits Zernike in 1953, and differential interference contrast (DIC) allow for the observation of cells and nuclei but also sub-cellular structures including microtubule bundles, chromosomes, nucleoli, organelle dynamics, and thick actin fibers12. Genetically encoded fluorescent proteins and the development of fluorescent dyes against organelles have dramatically impacted time-lapse microscopy13-15. While not the focus of this article, imaging in cell spheroids and in situ (intravital microscopy) using confocal and multiphoton microscopy represent another expansion of the approach, and there are outstanding articles that use and discuss these approaches16-19.
The responses of cells to anti-cancer drugs or natural products are determined on the molecular and cellular scale. Understanding cell responses and fates following treatment often involves population averaging assays (e.g., immunoblotting, whole well measures), or fixed time-points with immunofluorescent detection and flow cytometry, which measure single cells. Heterogeneity in single cell responses to drugs within a population, in particular in tumors, may explain some of the variability in response seen across cell lines and tumors that are treated with the same drug at saturation. Long-term longitudinal approaches to follow a given single cell or a population of cells is a less common but very powerful approach that allows for the direct study of molecular response pathways, different phenotypes (e.g., cell death or cell division), observation of cell-to-cell variability within a population, and how these factors contribute to population response dynamics20-22. Optimistically, being able to observe and quantify single cell responses will help improve our understanding of how drugs work, why they sometimes fail, and how to best use them.
The technique of long-term time-lapse microscopy, longitudinal tracking, and analysis of drug responses is available to many investigators and can be simple, using only transmitted light to observe phenotype responses20,21. The main components of the approach include: appropriate preparation of the cells of interest, an automated microscope with environmental chamber, a camera integrated with a computer to acquire and store the images, and software to review the time-lapse and measure and analyze the cells and any fluorescent biosensors. We provide a detailed protocol with many tips for conducting time-lapse microscopy of cultured cells for as long as several days using brightfield and/or widefield epifluorescent microscopy. This protocol can be used for any cell line that can be grown in culture to study their responses to anti-cancer therapies. We provide examples of data acquired and analyzed using multiple different genetically-encoded fluorescent biosensors and an example of phase-contrast microscopy, briefly discuss different types of probes, the advantages and disadvantages of long-term time-lapse and longitudinal tracking, what can be learned for this approach that is difficult to understand from non-direct approaches, and some variations that we hope will be of interest and value to inexperienced researchers who have not considered using the approach, and to experienced researchers.

<table><tbody><tr><td>Taxol (paclitaxel)</td><td>Sigma</td><td>T7191</td><td>microtubule stabilizing drug</td></tr><tr><td>Etoposide</td><td>Selleckchem</td><td>S1225</td><td>topoisomerase II inhibitor</td></tr><tr><td>Selinexor</td><td>Karyopharm Therapeutics</td><td>na</td><td>XPO1/CRM1 inhibitor, gift</td></tr><tr><td>Kinesin-5 inhibitor</td><td>Merck Serono</td><td>na</td><td>gift, also available from American Custom Chemicals Corporation. CAS 858668-07-2</td></tr><tr><td>Cell growth medium</td><td>HyClone (Fisher) or Mediatech</td><td></td><td>many companies available</td></tr><tr><td>5% CO2/balance air, certified</td><td>Airgas</td><td>Z03NI7222004379</td><td></td></tr><tr><td>35 mm Dish, 20 mm glass bottom</td><td>Cellvis</td><td>D35-20-1.5-N</td><td>many companies available</td></tr><tr><td>35 mm 4-well Dish, 20 mm glass bottom</td><td>Cellvis</td><td>D35C4-20-1.5-N</td><td>many companies available</td></tr><tr><td>35 mm Dish, gridded glass bottom</td><td>MatTek</td><td>P35G-2-14-CGRD</td><td>many companies available</td></tr><tr><td>Multi-well, glass bottom</td><td>Cellvis</td><td>P12-1.5H-N</td><td>many companies available</td></tr><tr><td>Olympus IX81 inverted epifluorescence microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Olympus IX2-UCB controller</td><td>Olympus</td><td></td><td></td></tr><tr><td>PRIOR LumenPro200</td><td>Prior Scientific</td><td>Lumen200PRO</td><td></td></tr><tr><td>PRIOR Proscan III motorized stage</td><td>Prio Scientific</td><td>H117</td><td></td></tr><tr><td>STEV chamber</td><td>InVivo Scientific</td><td>STEV.ECU.HC5 STAGE TOP</td><td></td></tr><tr><td>Environmental Controller Unit</td><td>InVivo Scientific</td><td>STEV.ECU.HC5 STAGE TOP</td><td></td></tr><tr><td>Hamamatsu ORCA R2 CCD with controller</td><td>Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>Nikon Eclipse Ti</td><td>Nikon</td><td></td><td></td></tr><tr><td>Nikon laser launch</td><td>Nikon</td><td></td><td></td></tr><tr><td>SOLA light engine</td><td>lumencor</td><td></td><td></td></tr><tr><td>iXon Ultra 897 EM-CCD</td><td>ANDOR&amp;nbsp;</td><td></td><td></td></tr><tr><td>TOKAI HIT inclubation chamber</td><td>TOKAI HIT</td><td>TIZSH</td><td></td></tr></tbody></table>

through the looking glass: time-lapse microscopy and longitudinal tracking of single cells to study anti-cancer therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Long-term time-lapse microscopy and direct longitudinal tracking allows for the study of many anti-cancer effects during drug response. Following the general outline in Figure 1, multiple examples of cells are shown expressing validated fluorescent reporters that treated with anti-cancer drugs, tracked, and analyzed using different approaches.
Phase contrast microscopy alone is very informative and robustly repo...

Watch this Scientific Journal Video about Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics at JoVE.com

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

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10.9K Views.  University of Colorado, Boulder. Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Video: Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

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 ...

Cancer Research

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 ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Biology

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Neuroscience

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to assess factors that influence neuronal fate. These assays require the observation of separate populations of cells at discrete time points. As a result, individual cells cannot be followed prospectively over time, severely limiting the ability to discriminate whether subcellular events, such as puncta formation or protein mislocalization, are pathogenic drivers of disease, homeostatic responses, or merely coincidental phenomena. Single-cell longitudinal microscopy overcomes these limitations, allowing the researcher to determine differences in survival between populations and draw causal relationships with enhanced sensitivity. This video guide will outline a representative workflow for experiments measuring single-cell survival of rat primary cortical neurons expressing a fluorescent protein marker. The viewer will learn how to achieve high-efficiency transfections, collect and process images enabling the prospective tracking of individual cells, and compare the relative survival of neuronal populations using Cox proportional hazards analysis.

Here, we present a protocol to monitor survival on a single-cell basis and identify variables that significantly predict cell death.

Standard cytotoxicity assays, which require the collection of lysates or fixed cells at multiple time points, have limited sensitivity and capacity to ...

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

Summary

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Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse Imaging of Mitosis After siRNA Transfection

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Monitoring Neuronal Survival via Longitudinal Fluorescence Microscopy