Name: live imaging to study microtubule dynamic instability in taxane-resistant breast cancers
Uploaded: 2017-02-20T13:00:00.000+00:00
Duration: 6 min 2 s
Description: Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

This research is supported by funding from CBCF (to ZW).

1. Preparing the Cells for Live Imaging
<ol>
	<li>Cell culture and seeding
	<ol>
		<li>Use MCF-7 breast cancer cells selected for resistance to docetaxel (MCF-7TXT) and their non-resistant parental cell line (MCF-7CC). The detailed selection process and the characterization of these selected cell lines were described previously24.</li>
		<li>Grow all cells in 10 cm culture dishes at 37 &#176;C in a medium composed 90% of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) and 10% of fetal bovine serum (FBS) and supplemented with non-essential amino acids. Maintain the medium in a 5% CO2 atmosphere. Maintain MCF-7TXT cells at 5 nM docetaxel.</li>
		<li>Seed cells on coverslips for live imaging. Sterilize 24 mm poly-L-lysine-coated glass coverslips by rinsing them with 70% alcohol and then exposing them to UV overnight. Place the coverslips into a 6-well culture plate of 35 mm. Place one coverslip in each well.</li>
		<li>Remove the medium from the 10-cm culture dish and wash the cells with PBS. Add 0.5 mL of trypsin-EDTA to the dish to detach the cells from the dish. Add 5 mL of culture medium to the dish to neutralize the trypsin following the detachment of the cells.</li>
		<li>Count the cells with a hemocytometer to determine the number of cell per unit volume.</li>
		<li>Add approximately 105 cells to each well based on the cell number per unit volume. After shaking it gently, put the plate back into the incubator to allow the cells to attach to the coverslips.</li>
	</ol>
	</li>
	<li>Expression of GFP-tagged &#945;-tubulin
	<ol>
		<li>To assay microtubule dynamics, transduce the cells with GFP-tagged &#945;-tubulin with the commercial GFP transduction control (e.g., BacMam) according to the manufacturer&#39;s protocol.</li>
		<li>Culture the cells in the well for 36 h in a 37 &#176;C cell culture incubator to allow complete adhesion and 60% confluence.</li>
		<li>Calculate the appropriate volume of GFP-tagged &#945;-tubulin to add to each well, according to the following equation: 
		<img alt="Equation" src="/files/ftp_upload/55027/55027eq1.jpg" /> 
		The number of cells is the estimated total number of cells in the well at the time of labeling and the desired PPC is the number of particles per cell. 
		&#8203;NOTE: Seeding the MCF-7 cells should have doubled the cell count from 105 to 2 x 105 with a desired PPC of 20, the volume needed for each coverslip is therefore 40 &#181;L. For different cells, the volume calculated based on the above formula may not be the best. The exact volume should be adjusted according to the actual expression level of GFP-tubulin.</li>
		<li>Mix the GFP-tagged &#945;-tubulin several times by inversion to ensure a homogenous solution. Do not vortex. 
		NOTE: For the control, the Null (control) reagent lacks any mammalian genetic elements and can be used to help determine potential baculovirus-mediated effects and background fluorescence. A non-targeted GFP transduction control may also be used, which will light up the entire cell.</li>
		<li>Replace the culture medium with fresh medium. Add 2 mL of the new medium and 40 &#181;L of GFP-tagged &#945;-tubulin to each well.</li>
		<li>Shake the plate gently to allow complete mixing of GFP-tagged &#945;-tubulin with the culture medium. Return the plate to the culture incubator and incubate for 24 h to allow the expression of GFP-tubulin.</li>
	</ol>
	</li>
	<li>Treat cells with docetaxel 
	NOTE: This experiment is to test the effect of docetaxel on microtubule dynamic instability. Thus, the cells are treated with the desired concentration of docetaxel.
	<ol>
		<li>Prepare a medium composed 90% of DMEM and 10% of FBS and supplemented with non-essential amino acids. Use DMEM without phenol red because phenol red may interfere with the fluorescence of GFP-tubulin. Add the desired concentration of docetaxel (ranging from 10 nM to 10 &#181;M). The stock solution of docetaxel is 10 mM in dimethyl sulfoxide (DMSO).</li>
		<li>Pre-warm the docetaxel-containing medium to 37 &#176;C in the tissue culture incubator.</li>
		<li>Replace the normal culture medium with the docetaxel-containing medium and incubate for 30 min in the tissue culture incubator. There should be at least 3 coverslips per well for each docetaxel concentration.</li>
	</ol>
	</li>
</ol>
2. Live Imaging to Examine Microtubule Dynamic Instability
<ol>
	<li>Setting up the system
	<ol>
		<li>Perform experiments in a chamber maintained at 37 &#176;C and 5% CO2. Acquire fluorescence images by time lapse with a microscopic system equipped with a 60X, 1.42 NA oil objective lens on an inverted fluorescence microscope and with a CCD camera.</li>
		<li>Set up the cells for live imaging. Pre-warm both the sample holder and the medium to 37 &#176;C. Choose a coverslip to examine. Mount the coverslip of interest on a sample holder and incubate it with 1 mL of the docetaxel-containing medium prepared in step 1.3.1.</li>
	</ol>
	</li>
	<li>Live imaging of microtubule dynamic instability
	<ol>
		<li>Identify the cells that will be used for imaging. They should be flat, have a high fluorescence intensity of expressed GFP-tubulin, and have clear microtubule structures.</li>
		<li>Choose one cell from the group of identified cells from step 2.2.1 to examine first. Focus on the peripheral area of the identified cell. Set up the program for exposure time and the frequency of image acquisition. As exposure time is usually 0.5 s, the images will be acquired every two s.</li>
		<li>Allow an additional 20 min for the steps from 1.33 to 2.2.3. Thus,exactly 50 min after the addition of docetaxel, as described in step 1.3.3, start imaging (this is for standardization).</li>
		<li>Record the microtubule dynamics for 2 min, taking a photo every 2 s. In total, acquire 60 images for the identified cell.</li>
		<li>Move to the next identified cell and repeat steps 2.2.2 to 2.2.4. Repeat the procedure until 5 cells have been imaged. It is imperative to do this before the time reaches 70 min after the addition of docetaxel, as described in step 1.3.3.</li>
		<li>For each treatment condition, repeat steps 2.2.1 to 2.2.5 for 3 different coverslips. In total, image 15 cells, which is enough data to measure microtubule dynamic instability.</li>
	</ol>
	</li>
</ol>
3. Image Processing and Data Analysis
<ol>
	<li>Deconvolution of images
	<ol>
		<li>Deconvolve the acquired images to achieve a high quality. Open the deconvolution program equipped with the microscope system.</li>
		<li>Click &#34;Process&#34; and select &#34;Deconvolve.&#34; Drag the image file to the &#34;Input&#34; window and click &#34;Do.&#34; The image file will be deconvolved, and the deconvolved file will be saved as a new file automatically.</li>
	</ol>
	</li>
	<li>Generation of videos
	<ol>
		<li>Click the deconvolved image file to open with equipped software.</li>
		<li>Click &#34;File&#34; and select &#34;Save as movie.&#34; Select the &#34;Movie format&#34; as &#34;AV&#34; and the &#34;Animation style&#34; as &#34;Forward.&#34; Set the &#34;Compression quality&#34; to &#34;100%&#34; and the &#34;Frame rate&#34; to &#34;5/second.&#34;</li>
		<li>Check the box of &#34;Animate through time&#34; and click &#34;Do it.&#34; The video will be generated.</li>
	</ol>
	</li>
	<li>Measurement of microtubule shortening and growth rate
	<ol>
		<li>Identify the microtubules that will be used for measurement. For each cell, only a few microtubules will be able to have their shortening or growth followed clearly and completely.</li>
		<li>Examine each frame of the video to identify the frame that shows the starting length of the microtubule and the frame with the most extended or shortened microtubule. Measure the change in length between the two microtubules and the time elapsed between the two frames.</li>
		<li>Calculate the rate of growth or shortening by dividing the change in length by time (the unit is &#181;m/s). For each treatment condition, follow at least 10 cells and measure at least 20 microtubules.</li>
		<li>Calculate the average and the standard error for each treatment condition. Analyze the statistical difference between the conditions using the Student test. Report the data in a table and/or in a chart.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

There are two major methods to measure microtubule dynamic instability: in vitro and in vivo. In the in vitro method, purified tubulin is used to measure microtubule dynamic instability with computer-enhanced time-lapse differential interference-contrast microscopy. In the in vivo method, microinjected fluorescent tubulin, or expressed GFP-tubulin, is incorporated into microtubules. The dynamics (growth and shortening) of the microtubules is then recorded by time-lapse using a sensitiv...

The leading cause of breast cancer mortality is through metastasis1,2. Taxanes, such as docetaxel and paclitaxel, are currently used as first-line regimens in the treatment of metastatic breast cancer2,3,4,5,6. They are part of a group of microtubule-targeting agents (MTAs) that disrupt microtubule dynamics. However, one of the greatest challenges to using taxanes in curative therapy is the development of taxane resistance in cancer cells, which leads to disease recurrence7. Drug resistance accounts for more than 90% of all deaths among patients with metastatic breast cancer7.
Microtubules are formed by the polymerization of &#945;- and &#946;-tubulin heterodimers8,9. The precise regulation of microtubule dynamics is important for many cellular functions, including cell polarization, cell cycle progression, intracellular transport, and cell signaling. Dysregulation of microtubules and their dynamics will disrupt cell function and result in cell death10,11. Depending on how they cause this dysregulation, MTA drugs can be classified as either microtubule stabilizing agents (i.e., taxanes) or microtubule-destabalizing agents (i.e., vinca alkaloids or colchicine-site binding agents)20. Despite their opposite effects on microtubule mass, at a sufficient dosage, both classes can kill cancer cells through their effects on microtubule dynamics21.
Taxanes function primarily by stabilizing the microtubule spindle12, leading to chromosomal misalignment. The subsequent perpetual activation of the spindle assembly checkpoint (SAC) arrests the cell in mitosis. Prolonged mitotic arrest then causes apoptosis13,14. Taxane interacts with microtubules through the taxane binding site on &#946;-tubulin8,15, which is only present in assembled tubulin16.
Multiple mechanisms for taxane resistance have been proposed9,17. These mechanisms include both general multidrug resistance due to the overexpression of drug-efflux proteins and taxane-specific resistance5,9,18,19. For example, taxane-resistant cancer cells may have altered expression and function of certain &#946;-tubulin isotypes5,9,19,20,21,22,23. By using an in vivo method to measure microtubule dynamic instability, we show that, when compared to non-resistant, parental MCF-7CC cells17, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment.
To better understand the function of MTAs and the exact mechanism of taxane-resistance in cancerous cells, it is essential to measure microtubule dynamics. Here, we report an in vivo method of doing so. By using live imaging in combination with the expression of GFP-tagged tubulin in cells, we can measure the microtubule dynamics of MCF-7TXT and MCF-7CC cells with and without docetaxel treatment. The results can help us design more effective drugs that can overcome taxane resistance.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Life Technologies, Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>FBS</td><td>Gibco, Invitrogen</td><td>12483</td><td></td></tr><tr><td>Anti-Anti (100x)</td><td>Life Technologies, Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>docetaxel</td><td>Sigma-Aldrich</td><td>01885-5mg-F</td><td></td></tr><tr><td>DMEM phenol red-free</td><td>Gibco, Invitrogen</td><td>21063</td><td></td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td><td>ThermoFisher Scientific</td><td>C10613</td><td>Key reagent for expressing GFP tubulin in cells</td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP</td><td>ThermoFisher Scientific</td><td>B10383</td><td>Control</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich+B9:AA9</td><td>472301</td><td>for dissoving decetaxel</td></tr><tr><td>22-mm glass coveslip</td><td>Fisher Scientifics</td><td>12-545-101</td><td></td></tr><tr><td>6-well culture plate</td><td>Greiner Bio-One International</td><td>6 Well Celi Culture Plate</td><td></td></tr><tr><td>DeltaVision Microscopy Imaging Systems</td><td>GE Health</td><td></td><td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Bright-Line Hemacytometer Set, Hausser Scientific</td><td>Hausser Scientific, Distributed by VWR</td><td>Supplier No.: 1492 VWR No.:15170-172</td><td></td></tr></tbody></table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

Using the protocol presented here, we studied the effects of docetaxel on the microtubule dynamics of normal (MCF-7CC) and docetaxel-resistant (MCF-7TXT) breast cancer cells. Two sets of images show the effects of docetaxel (0.5 &#181;M) on microtubule growth and shortening in MCF-7CC and MCF-7TXT cells (Figure 1A).
We also calculated the rate of microtubule growth or ...

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Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

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7.5K Views.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

Video: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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 ...

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunology and Infection

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Biochemistry

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.

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 ...

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 to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

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Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

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

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

Real-time Imaging of Myeloid Cells Dynamics in Apc^Min/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

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

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