We would like to acknowledge our funding support by the Bloomfield Group Foundation through the Hunter Medical Research Institute (HMRI 13-02). X.Z is supported by an APA scholarship through the University of Newcastle and the HCRA Biomarkers Flagship PhD Scholarship.

NOTE: Two cell lines should be handled separately. The following procedures should be applied to one single cell line if not specified.
1. Optimize Cell Density Prior to Wounding
<ol>
	<li>Culture adherent cells in T75 cm2 tissue culture flasks to about 80% confluence in phenol-red free Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine and 2 &#181;g/mL insulin at 37 &#176;C with 5% CO2 (standard incubation conditions for most cancer cell lines, culture formulations are cell line-dependent).</li>
	<li>Remove the culture media by pipetting off into a waste container. Pipet 2 mL of pre-warmed 0.1% trypsin-EDTA to briefly rinse the cell monolayer and pipet off. Add another 2 mL of 0.1% trypsin-EDTA and place the flask under standard incubation conditions at 37 &#176;C for 5 min.</li>
	<li>Gently tap the flask to ensure detachment of the cells and then add 10 mL of pre-warmed culture media to stop the proteolytic reaction.</li>
	<li>Transfer the cell suspension into a 15 mL centrifuge tube and spin down at 200 x g for 5 min at room temperature (RT). Carefully remove the supernatant without agitating the cell pellet and resuspend with another 10 mL pre-warmed culture media.</li>
</ol>
2. Counting Cell Number Using an Automated Cell Counter (or Any Counting Methods)
<ol>
	<li>Dilute 200 &#181;L of resuspended cell suspension with 800 &#181;L of 1x Dulbecco&#39;s Phosphate buffered Saline (DPBS) in a 1.5 mL centrifuge tube.</li>
	<li>Attach a 60 &#181;m cell count sensor to the cell counter, hold down the toggle and merge the tip into the cell suspension. Slowly release the toggle until the cell suspension is successfully drawn into the sensor. Cell concentration is displayed in cells/mL.</li>
	<li>Calculate the cell number in the 10 mL cell suspension.</li>
</ol>
3. Cell Plating
<ol>
	<li>Plate cells at a range of cell densities (40,000-90,000 cells/well) in triplicate in a 96-well plate.</li>
	<li>Place the plate into the live-cell imager, and schedule scanning every 2 h for 24 h.
	<ol>
		<li>In the software, click Schedule scans from the task list. In the drawer setup pane, determine the position of the plate, click Add vessel and choose the plate type. In the Scan Setup pane, choose or edit the scan pattern according to the experimental plate setup, and set Scan Type as Standard.</li>
		<li>Right click on the Timeline and select Set Intervals. Set Add Scans Every to 2 h at a 24-h schedule. Click Apply.</li>
	</ol>
	</li>
</ol>
4. Determine the Optimal Cell Seeding Density for the Migration Assay
<ol>
	<li>Stop scanning after 24 h, apply the confluence processing analysis tool to the HD-phase contrast images automatically collected, and generate a cell proliferation curve against time.
	<ol>
		<li>Determine 3-6 representative images and place them in a new Image Collection.</li>
		<li>Determine a proper mask as Processing Definition.</li>
		<li>Launch an analysis job.</li>
		<li>Determine optimized cell density according to confluence against time (approximately 100% confluence within 6 -18 h depending on when the migration assay commences). 
		NOTE: The amount of time it takes to grow the cells to confluence varies depending on the seeding dilution.</li>
	</ol>
	</li>
</ol>
5. Days 1 and 2: Preparation for Migration and Invasion Assays
<ol>
	<li>On Day 1, coat the plate for invasion assay. 
	NOTE: ECM gel should always be handled on ice and with tips that have been placed in the fridge overnight.
	<ol>
		<li>Dilute ECM gel with ice-cold culture media to 100 &#181;g/mL and add 50 &#181;L of diluted ECM gel/media into designated wells.</li>
		<li>Place the plate at standard incubation conditions overnight.</li>
	</ol>
	</li>
	<li>On day 2, gently aspirate the excess media. Plate cells with optimized cell densities into 2 96-well plates designated for the migration assay (uncoated) and the invasion assay (coated) in triplicates following sections 1-3 in the late afternoon (in the current protocol, optimized seeding densities for ZR75-1 and MDA-MB-231 were 90,000/well and 50,000/well, respectively).</li>
	<li>Place plates at standard incubation conditions overnight.</li>
</ol>
6. Day 3: Wound Scratch
<ol>
	<li>Spray and wipe the scratch tool and 2 washing boats with 70% ethanol before placing them in the biosafety cabinet. Fill the washing boat 1 and 2 with exactly 45 mL of sterile (autoclaved) distilled water and 70% ethanol respectively.</li>
	<li>For sterilization, place the scratch tool pin block (top) on washing boat 1 and 2 for 5 min each.</li>
	<li>Start with the migration assay plate.
	<ol>
		<li>Move the plate containing cells from the incubator and make sure no well is dry to avoid damage of the scratch tool. Remove the plate cover and insert into the base plate holder of the scratch tool, and carefully place the top part onto the base part by guiding dowels. Press and hold the black lever, and meanwhile carefully lift the pin block. Gaps in each well are usually visible with the naked eye and under the microscope.</li>
		<li>Quickly soak the pins in water; this is sufficient to clean the scratch tool prior to scratching the invasion assay plate if plate setup is identical. Otherwise, repeat the sterilization steps with sterile distilled water and then 70% ethanol for 5 min each.</li>
	</ol>
	</li>
	<li>Wash the plate 1 or 2 times with pre-warmed culture media to avoid detached cells or cell sheets reattaching to the well.</li>
	<li>Add 100 &#181;L of fresh warm media into designated wells with or without treatments.</li>
	<li>Additional steps for invasion assay.
	<ol>
		<li>Place the invasion assay plate at 4 &#176;C for 5 min to equilibrate and carefully aspirate the cold media.</li>
		<li>Dilute ECM gel with ice-cold culture media to 5 mg/mL, add 50 &#181;L of diluted ECM gel into designated wells, and place under standard incubation for 30 min.</li>
		<li>Add 100 &#181;L of warmed-up media with or without testing compounds.</li>
	</ol>
	</li>
	<li>Place the plate into live-cell imager and let it equilibrate for 5 min. Choose Vessel Type as imagelock plate. Set Scan Type as Scratch Wound, choose or edit Scan Pattern according to the experimental plate setup (1 image/well and wide mode) and schedule 24-h repeat scanning every 1-2 h for 72 h until the wounds are healed.</li>
	<li>To clean the scratch tool, put the top pin block in each of the following solutions (45 mL in washing boats) for 5 min: 0.5% detergent 1 (see Table of Materials), 1% detergent 2 (see Table of Materials), sterile distilled water and 70% ethanol. Place the scratch tool back onto its base plate and store in a dust-free environment.</li>
</ol>
7. Data Analysis
<ol>
	<li>Stop scanning the designated plate after all the wounds have healed by choosing the experimental plate on the Drawer Setup and clicking Remove Vessel.</li>
	<li>Collect 3-6 representative images spanning a range of sound percentages, including images right after the wound has been made, wound closure by 10% and 50%.</li>
	<li>To determine a proper processing definition, use Segmentation Adjustment, Cleanup and Filters to apply appropriate Scratch Wound Mask and Confluence Mask. Use Preview current/all to view the accuracy of the masks.</li>
	<li>Launch the analysis job.</li>
	<li>Data can be analyzed within the software or exported for further analysis. Three metrics provided by the software can be used to evaluate the HD-phase images: wound width (&#181;m), wound confluence (%) and relative wound density (%). Comparisons will be discussed in the following section.</li>
</ol>

<ol>
	<li>Graham, C. H., Lala, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+placental+invasion+of+the+uterus+and+their+control.">Mechanisms of placental invasion of the uterus and their control.</a> Biochemistry and Cell Biology. 70, 867-874 (1992).</li><li>Affolter, M., Zeller, R., Caussinus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+remodelling+through+branching+morphogenesis.">Tissue remodelling through branching morphogenesis.</a> Nature Reviews Molecular Cell Biology. 10, 831-842 (2009).</li><li>Brugues, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forces+driving+epithelial+wound+healing.">Forces driving epithelial wound healing.</a> Nature Physics. 10, 683-690 (2014).</li><li>Weigelt, B., Peterse, J. L., van't Veer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+metastasis:+markers+and+models.">Breast cancer metastasis: markers and models.</a> Nature Reviews Cancer. 5, 591-602 (2005).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> Journal of Clinical Investigation. 119, 1420-1428 (2009).</li><li>Scully, O. J., Bay, B. H., Yip, G., Yu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+metastasis.">Breast cancer metastasis.</a> Cancer Genomics Proteomics. 9, 311-320 (2012).</li><li>Kramer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+cell+migration+and+invasion+assays.">In vitro cell migration and invasion assays.</a> Mutation Research/Reviews in Mutation Research. 752, 10-24 (2013).</li><li>Holliday, D. L., Speirs, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+the+right+cell+line+for+breast+cancer+research.">Choosing the right cell line for breast cancer research.</a> Breast Cancer Research. 13, 215 (2011).</li><li>Clark, A. G., Vignjevic, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modes+of+cancer+cell+invasion+and+the+role+of+the+microenvironment.">Modes of cancer cell invasion and the role of the microenvironment.</a> Current Opinion in Cell Biology. 36, 13-22 (2015).</li></ol>

The authors declare that they have no competing financial interests.

Migration and invasion are important parameters to assess the mobility of cancer cells. By using the 96-pin scratch tool, it is possible to conduct wound healing assays in 2D and 3D simultaneously. Apart from facilitating automatic scanning, providing a stable cell culture environment with minimum disruption, the scratch assay conducted using the 96-pin scratch tool provides consistent scratch wounds, enabling experiments that are more robust and reproducible. The 96-well plate format gives additional options of either i...

Cell migration and invasion are important biological processes that enable normal functions in the human body, such as wound closure, invasion of placenta into the uterus and mammary gland morphogenesis1,2,3. The human body has precise and strict control of these biological events; however, there are some exceptions. Malignant tumors, for example, are able to escape this safeguard, exhibit abnormal proliferation and invade into neighboring tissue, which is called metastasis. Metastasis is the major cause of cancer-related mortality4.
Breast cancer is the most commonly diagnosed cancer in women, and is the second-highest cause of cancer-related death among women in developed countries worldwide5. Breast cancer originates from ducts or lobules that consist of one or more layers of epithelial cells. In the normal breast, epithelial cells adhere to one another and to the basement membrane through membrane proteins such as E-cadherin and integrins6. However, invasive breast cancer cells have lost their polarity and cell-cell adhesion, and classically undergo epithelial mesenchymal transition (EMT) and gain the ability to move. After extravasation, these cells move across the extracellular matrix (ECM) and enter the blood vessel or the lymphatic system, followed then by intravasation and metastatic growth7. Understanding the mechanisms by which this occurs is of great significance, since metastasis is the most common cause of cancer-related mortalities and is closely related to cancer cell migration/invasion. To visualize the movement of cancer cells, migration and invasion assays are ideal models to study 2D and 3D cell movement, respectively. Migration directly assesses the movement of the cells whereas invasion involves interaction with the microenvironment and the ability to degrade biological barriers. The two processes are not fully independent of one another, as migration is a requirement of invasion.
Several methods have been developed to study migration and invasion. As reviewed by Kramer et al., migration assays such as wound healing, fence and micro-carrier assays generate a cell-free area to allow cells to move into, assessing the change of area; whereas, transwell and capillary assays are based on the number of cells that move toward an attractant8. For invasion assays, an ECM environment has to be set up with ECM gel or collagen for instance, and 3D movement can be assessed by monitoring the invasion area, distance and cell counts (e.g. transwell assay, platypus assay)8. Another type of invasion assay is to combine the invasive cells with non-invasive cells and assess the behavior of the invasive cells (e.g. spheroid assays). The above methods have their pros and cons, and a way that is easy to approach, easy to repeat, and to combine the migration assay and invasion assay in a similar workflow is preferential in experimental design.
This protocol describes the measurement of cell migration and invasion using a live-cell imager. It is a real-time cell monitoring system installed in a standard cell culture incubator. It takes high definition images according to the set scanning intervals and measurements by applying appropriate masks to the cells or fluorescent targets. The module of migration/invasion assay includes using a 96-pin scratch tool, which is suitable for making homogeneous scratch wounds on a cell monolayer in a 96-well plate. The mechanism is based on in vitro wound healing assays, monitoring 2D cell movement on a plastic or coated surface. Invasion or 3D movement across an additional ECM within the scratch wound can also be assessed. A brief workflow is illustrated in Figure 1.

<table border="0" cellpadding="0" cellspacing="0" style="width:695px;" width="695">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">0.5% trypsin-EDTA solution (10x)</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">30028-02</td>
			<td style="width:167px;">Dilute to 2x in DPBS</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Countess II FL Automated Cell Counter</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AMQAX1000</td>
			<td style="width:167px;">Automated cell counter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Detergent 1 (Alconox)</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">242985</td>
			<td style="width:167px;">0.5% working concentration</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Detergent 2 (Virkon S)</td>
			<td style="width:100px;">VetProduct DIRECT</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1% working concentration</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:309px;">Dulbecco&#8217;s Modified Eagle Medium, no phenol-red</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">21063-045</td>
			<td style="width:167px;">Supplimented with 10% FBS, 200 mM L-glutamine, 2 &#956;g/ml insulin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">ECM Gel (matrigel)</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">E6909</td>
			<td style="width:167px;">Growth-factor reduced, phenol red free</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Essen ImageLock 96-well plate, flat bottom</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;">4379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">EVE Counting slides</td>
			<td style="width:100px;">BioTools</td>
			<td style="width:119px;">EVS-50</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Fetal bovine serum (FBS)</td>
			<td style="width:100px;">Bovogen Biologicals</td>
			<td style="width:119px;">SFBS-F-500ml</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">IncuCyte 96-well scratch wound cell invasion accessories</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;">4444</td>
			<td style="width:167px;">Including CoolBox, 2x CoolSink</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:309px;">IncuCyte Cell migration kit</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;">4493</td>
			<td style="width:167px;">Including the 96-well pin block, 2x wash boats and the software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">IncuCyte ZOOM</td>
			<td style="width:100px;">Essen</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Live cell analysis system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:309px;">Insulin solution human</td>
			<td style="width:100px;">Sigma-Aldrich</td>
			<td style="width:119px;">19278-5ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">L-glutamine solution (100x)</td>
			<td style="width:100px;">ThermoFisher Scientific</td>
			<td style="width:119px;">25030-091</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:309px;">Tissue culture flask, 75 cm2 growth area</td>
			<td style="width:100px;">Greiner Bio-One</td>
			<td style="width:119px;">658175</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a simple migration/invasion workflow using an automated live-cell imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

This migration/invasion assay is based on the wound healing assay, which evaluates the rate of the cells moving into a cell-free area created by the 96-pin scratch tool. The difference between the migration and invasion assays are that migration assays measure cells moving on the tissue-culture treated plastic surface and invasion measures cells moving across ECM gel.
The scratch tool is designed to make consistent scratch wound...

Watch this Scientific Journal Video about A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager at JoVE.com

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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7.6K Views.  Hunter Medical Research Institute. The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Video: A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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

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

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

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

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

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

A Data Integration Workflow to Identify Drug Combinations Targeting Synthetic Lethal Interactions

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of both synthetic lethal interactors leads to loss of cell viability or cell death. The best studied synthetic lethal interaction is between BRCA1/2 and PARP1, with PARP1 inhibitors being used in clinical practice to treat patients with BRCA1/2 mutated tumors. Large genetic screens in model organisms but also in haploid human cell lines have led to the identification of numerous additional synthetic lethal interaction pairs, all being potential targets of interest in the development of novel tumor therapies. One approach is to therapeutically target genes with a synthetic lethal interactor that is mutated or significantly downregulated in the tumor of interest. A second approach is to formulate drug combinations addressing synthetic lethal interactions. In this article, we outline a data integration workflow to evaluate and identify drug combinations targeting synthetic lethal interactions. We make use of available datasets on synthetic lethal interaction pairs, homology mapping resources, drug-target links from dedicated databases, as well as information on drugs being investigated in clinical trials in the disease area of interest. We further highlight key findings of two recent studies of our group on drug combination assessment in the context of ovarian and breast cancer.

Large genetic screens in model organisms have led to the identification of negative genetic interactions. Here, we describe a data integration workflow using data from genetic screens in model organisms to delineate drug combinations targeting synthetic lethal interactions in cancer.

A synthetic lethal interaction between two genes is given when knock-out of either one of the two genes does not affect cell viability but knock-out of ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in glioblastoma (GBM), the most prevalent and devastating primary brain tumor. The presence of GSCs makes the GBM very refractory to most of individual targeted agents, so high-throughput screening methods are required to identify potential effective combination therapeutics. The protocol describes a simple workflow to enable rapid screening for potential combination therapy with synergistic interaction. The general steps of this workflow consist of establishing luciferase-tagged GSCs, preparing matrigel coated plates, combination drug screening, analyzing, and validating the results.

The glioma stem cells (GSCs) are a small fraction of cancer cells which play essential roles in tumor initiation, angiogenesis, and drug resistance in ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

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

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

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

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