This work was supported by grants from the National Health and Medical Research Council (NHMRC) to H-JZ. TMBW is a recipient of an Australia Postgraduate Award from the Australian Government and the Ann Henderson Top-Up Scholarship from Australian Rotary Health in partner with Rotary of Templestowe and Dine for a Cure.

All animal experiments were approved by the University of Melbourne Animal Ethics Committee.
Note: The sequence, construction and generation protocol for the adenoviral vectors pAd-CMV-Td-Tom, pAd-CMV-GFP and pAd-CAGA12-Luc/Td-Tom have been previously described11,12,13. All vectors are commercially available.
1. Virus Titer Determination Using 50% Tissue-culture Infectious Dose (TCID50)
<ol>
	<li>Prepare 1 x 106 HEK293A cells in 10 mL of DMEM + 10% FBS media.</li>
	<li>Seed 100 &#181;L of cell suspension into each well of a 96-well flat-bottom cell culture plate.</li>
	<li>Prepare 1 mL of a 1:100 dilution of virus stock in complete medium. Add 11 &#181;L of diluted virus to each well of column 1.</li>
	<li>Perform serial 10-fold dilutions directly in the 96 well plate by mixing 11 &#181;L of diluted virus with 100 &#181;L of cell suspension until a final dilution of 1 x1012 is reached. Although cells are non-adherent at this stage, the number of cells transferred between wells remains constant.</li>
	<li>Pipette 11 &#181;L of each dilution into each column starting with the highest dilution of 104. Replace pipette tips with each dilution to limit cross-over of viral particles.</li>
	<li>Add 11 &#181;L of virus-free complete media to column 12 as a negative control.</li>
	<li>Incubate plate for 7 - 10 days at 37 &#176;C with 10% CO2.</li>
	<li>Using a microscope, score the number of wells in each column displaying signs of cytopathic or cytotoxic effects. Consider a well positive if any sign of infection is present.</li>
	<li>Calculate the fraction of positive wells for each dilution using the Reed and Muench (1938) statistical method14.</li>
	<li>Calculate the proportional distance (PD) using: (A-50)/(A-B), where A is the percentage response above or equal to 50% and B is the percentage response below 50%.</li>
	<li>Calculate TCID50 using: 10X-PD, where X is the dilution giving a response greater than 50%.</li>
	<li>Calculate viral titer from using the following: 0.69/(0.011 x TCID50) PFU/mL</li>
</ol>
2. Adenovirus MOI Determination
<ol>
	<li>Seed 1.0 - 1.5 x 105 MDA-MB-231 cells with 500 &#956;L of DMEM culture media containing 5% FBS in each well of a 12 well plate (35% cell confluency, 6 wells in total).</li>
	<li>Calculate the volumes of Ad-CMV-Td-Tom stock required for 0, 250, 500, 2500, and 5000 MOI using following formula: MOI = (volume [&#956;L] x PFU/&#956;L)/number of cells. For Ad-CMV-Td-Tom with a titer of 1 x 1010 (PFU/mL), the volumes of virus infection required MOIs are 0, 3.75, 7.5, 37.5, and 75 &#956;L respectively.</li>
	<li>Infect the cells with the calculated volumes of Ad-CMV-Td-Tom in Section 2.2 by directly pipetting viral stock into the wells with thorough mixing.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Remove the media from the wells and immediately fix the cells with 500 &#956;L of 10% formalin for 5 min. &#160;</li>
	<li>Aspirate the formalin from wells and wash wells with 500 &#956;L of PBS once.</li>
	<li>Stain the cell nucleus with 500 &#956;L of Hoechst solution for 5 min.</li>
	<li>Remove the Hoechst solution and wash the wells with 500 &#956;L of PBS 3 times.</li>
	<li>Image the Td-Tomato protein and cell nucleus signal using 200X magnification on a fluorescence microscope. Excite Td-Tomato protein at 554 nm and the Hoechst dye at 352 nm.</li>
	<li>Analyze images using ImageJ to determine the working MOI required for 100% infection of Td-Tomato15.</li>
</ol>
3. Dual-adenovirus Transduction in Live Single Cells
<ol>
	<li>Seed 1.0 - 1.5 x 105 MDA-MB-231 cells with 500 &#956;L of DMEM culture media containing 5% FBS in each well of a 12 well plate (35% cell confluency, 2 wells in total).</li>
	<li>Infect cells with 75 &#956;L of Ad-CMV-GFP (titer = 5 x 109 PFU/mL) and 7.5 &#956;L of Ad-CAGA12-Td-Tom (titer = 5 x 1010 PFU/mL) to obtain a 2,500 MOI, which can achieve 100% infection positivity.</li>
	<li>Treat cells with or without 0.25 &#956;L of TGF-&#946; per well (10 mg/mL in stock, 5 ng/&#956;L as the final concentration) immediately after adenovirus infection.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Image live cells with phase contrast fluorescence microscope. Excite GFP at 470 nm and Td-Tomato protein at 554 nm.</li>
	<li>Fix cells and stain cell nucleus as described in Steps 1.6 - 1.9.</li>
	<li>Image the Td-Tomato protein and Hoechst stain using a fluorescence microscope. Excite the Hoechst dye at 352 nm.</li>
	<li>Using ImageJ, calculate the percentage of GFP and Td-Tomato positive cells15.</li>
</ol>
4. Live Single Cell Signaling in Wound Healing Assay
<ol>
	<li>Seed 4 - 5 x 105 MDA-MB-231 cells with 1 mL of DMEM culture media containing 5% FBS in each well of a 6 well plate (50% cell confluency, 2 wells in total).</li>
	<li>Infect the cells with 22.5 &#956;L of Ad-CAGA12-Td-Tom (titer = 5 x 1010 PFU/mL, MOI = 2,500).</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Scratch two crossed lines on the cell layer of each well using a P200 pipette tip.</li>
	<li>Remove the old media from wells and replace with 2 mL of fresh 5% FBS media with or without 0.5 &#956;L of TGF-&#946; per well (10 mg/mL in stock, 5 ng/&#956;L as final concentration).</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 5 min and take images of selected wound areas using 100X magnification on a phase contrast microscope.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Take images of the wound closure at the same areas as before.</li>
	<li>Fix the cells, stain cell nucleus as described in Steps 1.6 - 1.9 and visualize Td-Tomato signal in the wound area and non-wound area using 200X magnification on a fluorescence microscope.</li>
	<li>Quantify the percentage of Td-Tomato positive cells in the wound and non-wound area using ImageJ software15.</li>
</ol>
5. In Vitro Luciferase Assay
<ol>
	<li>Prepare a cell suspension of 3 - 5 x 104 MDA-MB-231 cells in 1 mL of DMEM media containing 5% FBS.</li>
	<li>Infect cell suspension with 2.5 &#956;L of Ad-CAGA12-Luc (titer = 5 x 1010 PFU/mL, MOI = 2,500).</li>
	<li>Seed the infected cells into a 96 well plate at 3,000 cells/100 &#181;L/well.</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Remove old media from wells and replace with 100 &#181;L of fresh 5% FBS media with or without 0.1 &#956;L of TGF-&#946; per well (1 mg/mL in stock, 1 ng/&#956;L as the final concentration) and in the presence or absence of 0.17 &#956;L of TGF-&#946; inhibitor per well (7 mg/mL in stock, 12 ng/&#956;L as final concentration) (a novel TGF-&#946; ligand trap protein, Chen et al., unpublished work).</li>
	<li>Place the plate in the incubator at 37 &#176;C with 10% CO2 for 24 h.</li>
	<li>Thaw out the luciferase assay system substrate and prepare 1x cell culture lysis reagent in double distilled water (DDW).</li>
	<li>Remove media from the 96 wells and lyse wells with 50 &#956;L of 1x cell culture lysis buffer on ice using an orbital shaker with gentle agitation for 30 min.</li>
	<li>Transfer 30 &#956;L of lysate from each well into an opaque luciferase plate and allow the plate to warm to room temperature.</li>
	<li>On the luminometer software, create a new protocol. Select for 1 injector with measurements before and after injection. Set measurements before injection at 1 s for both Delay in Measurement and Integration Time. For after injection measurement, set the Injection Volume to 40 &#181;L with a Delay in Measurement of 1 s after injection and 5 s Integration Time. Confirm settings by selecting OK.</li>
	<li>Place the injector tube into DDW and click the Injector 1 button under the Prime settings to clean the injector tube before use. Next remove the injector tube from the DDW and place in air followed by two further Primes of injector 1 to flush all solution from the tube. Place the injector tube into Firefly substrate and again Prime twice to prepare the substrate.</li>
	<li>Place the opaque luciferase plate with the samples into the luminometer and select Options on the software. Highlight the wells of interest and click Apply. Press Start to measure luciferase signal.</li>
	<li>Once measurements are complete, remove and rinse the plate with water. Recover any remaining substrate into the injector tube by placing the tube into air and Prime injector 1 back into firefly substrate tube. Place the injector tube into DDW and perform two further Primes to clean the injector tube of any remaining substrate.</li>
</ol>
6. Cell Preparation for Live Signaling Imaging In Vivo
<ol>
	<li>48 h before tumor implantation, seed 2 - 3x106 MDA-MB-231 cells into 175 cm2 flasks (10 flasks in total). Culture cells in 20 mL of DMEM media containing 5% FBS at 37 &#176;C, 10% CO2.</li>
	<li>24 h later, add 300 &#956;L of Ad-CAGA12-Luc (titer = 5 x 1010 PFU/mL, MOI = 2,500) into each flask. Keep cells in culture for another 24 h.</li>
	<li>On the day of implantation, remove the old media and wash the adenovirus infected cells once with 20 mL of PBS, pH 7.4. Add 2 mL of 0.05% trypsin-EDTA into the flask and shake the flask slightly to allow trypsin to cover all the flask surface. Place the flask in to the incubator 37 &#176;C, 10% CO2 for 3 min.</li>
	<li>Quench the trypsin by adding 8 mL of FBS-containing DMEM media into flasks. Transfer all cell suspensions into a glass reagent bottle.</li>
	<li>Count the cell number using a hemocytometer and transfer 4.8 x 107 cells in two 50ml centrifuge tubes.</li>
	<li>Centrifuge the cells at 400 x g for 5 min at RT to remove trypsin and resuspend cells in 720 mL of fresh FBS-DMEM media.</li>
	<li>Transfer the cell suspensions into a sterile 5 mL tube and add 160&#160;&#956;L Matrigel (10% total matrigel suspension).</li>
	<li>Keep cells on ice until implantation.</li>
</ol>
7. Orthotopic Implantation
<ol>
	<li>Weigh 12 SCID mice and randomly allocate mice into control and treatment groups (6 mice/group).</li>
	<li>Perform the implantation in a sterile bio-cabinet to maintain a sterile environment. Anaesthetize the mouse using 2.5 -&#160;4.5% isoflurane with an oxygen flow rate of 1 L/min. Place the mouse on a heat pad and dispense a drop of lubricating eye ointment onto both eyes to avoid corneal damage. Fix the mouse to the heat pad in a supine position while maintaining anaesthesia by placing the snout into a nose cone connected to the isoflurane.</li>
	<li>Confirm the success of anaesthesia by the lack of reaction to toe pinch. Reduce isoflurane to a maintenance dose of 1.5 -&#160;2.5% with 1 L/min oxygen for the reminder of implantation.</li>
	<li>Clean the skin from the fourth nipple on both sides to the midline by using the cotton swab dipped into 80% ethanol.</li>
	<li>Find the 4th mammary fat pad through palpation and squeeze the fat pad to further expose the tissue.</li>
	<li>Mix the cell suspension well by pipetting up and down. Gently aspirate 50 &#181;L of cell mixture into a 27G insulin syringe and inject into the mammary fat pad on both sides of the mouse.</li>
	<li>Confirm a successful injection by feeling for swelling of the fat pad. Release the fat pad gently and place the mouse back into the cage.</li>
	<li>Leave the cages on the heat pad for at least 30 min to allow the mice to gain consciousness and then place the cages back in the holding room for 3 days.</li>
</ol>
8. IVIS Imaging
<ol>
	<li>Open the Living Image software.</li>
	<li>Initialize the IVIS system by clicking the Initialize IVIS System button on the control panel. Wait until the temperature sign turns green.</li>
	<li>Select the Luminescent Imaging Mode on the control panel.</li>
	<li>Turn on isoflurane anaesthesia to the chamber and IVIS system.</li>
	<li>Anaesthetize the mice by using 2.5 -&#160;4.5% isoflurane with 1 L/min oxygen. Once anaesthetized, set isoflurane at 1.5 -&#160;2.5% for maintenance. Inject 150 mg/kg body weight of D-luciferin into mice by intraperitoneal (i.p.) injection.</li>
	<li>Immediately transfer mice into the IVIS system, place them onto the temperature-controlled imaging platform and place their nose into the nose cone.</li>
	<li>Click on the Sequence Setup button on the control panel and select Medium Binning.</li>
	<li>Set up the imaging exposure time as follows: 10 s, 20 s, 30 s, 60 s, and 120 s. Start imaging approximately 3 min after the luciferin injection and continue to image until the signal starts to drop (normally this will take around 20 min).</li>
	<li>Remove the mice from IVIS while maintaining isoflurane anaesthetic through a nose cone and treat the mice with 50 &#181;L PBS or 50 &#181;L TGF-&#946; signaling inhibitor (50 &#956;g/tumor) via intra-tumor injection.</li>
	<li>Put the mice back in the cage and leave them on the heat pad for at least 30 min. Then place the cage back in the holding room.</li>
	<li>Repeat IVIS imaging procedure as described above the day following treatment.</li>
</ol>
9. Data Analysis
<ol>
	<li>Open saved files in Living Image software.</li>
	<li>Find the image information using View | Image Information.</li>
	<li>Select photos taken at a similar time point with the same exposure time. Load photos as a group.</li>
	<li>Uncheck the Individual box in Image Adjust to normalize the intensity.</li>
	<li>Select Photon mode to analyze the intensity. Quantify the intensity of the signal by selecting the Region of Interest (ROI) button in ROI Tools.</li>
	<li>Drag the ROI circle to the region containing the bioluminescent signal and click the Measure button to acquire the signal intensity.</li>
</ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+adenovirus+vectors+with+modified+fibers+for+altering+viral+tropism.">Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.</a> Journal of Virology. 70, 6839-6846 (1996).</li><li>Wickham, T. J., 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=Increased+in+vitro+and+in+vivo+gene+transfer+by+adenovirus+vectors+containing+chimeric+fiber+proteins.">Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.</a> Journal of Virology. 71, 8221-8229 (1997).</li><li>Smith, F., Jacoby, D., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+vectors+for+gene+delivery+to+the+nervous+system.">Virus vectors for gene delivery to the nervous system.</a> Restorative neurology and neuroscience. 8, 21-34 (1995).</li><li>Zhou, F., 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=Nuclear+receptor+NR4A1+promotes+breast+cancer+invasion+and+metastasis+by+activating+TGF-beta+signalling.">Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-beta signalling.</a> Nature communications. 5, 3388 (2014).</li></ol>

The authors report no conflicts of interest.

We have developed a technique to allow for real-time imaging of TGF-&#946;/Smad3 signaling in single live cells. Using this novel method, we have previously identified a sub-population of cells with dynamic TGF-&#946;/Smad3 transcriptional activity that was associated with enhanced invasion and migration8. This method improves on traditional assays for TGF-&#946; signaling, such as western blots of Smad3 phosphorylation and TGF-&#946; targeted gene expression, by capturing the heterogeneity of TGF...

Transforming Growth Factor &#946; (TGF-&#946;) is an essential cytokine implicated in human development that signals through a heterodimeric complex consisting of type II and type I receptors1. Binding to type II receptor results in the recruitment and phosphorylation of type I receptor, which in turn phosphorylates downstream Smad2/3 proteins2,3. These activated Smad2/3 proteins bind to Smad4, forming a complex that translocates into the nucleus and regulates gene transcription4. Under homeostatic conditions TGF-&#946;/Smad signaling is tightly regulated; however, in many diseases, the signaling pathway is deregulated and often overexpressed leading to progression of disease5,6,7. Recent studies have demonstrated that cellular response to TGF-&#946; is heterogenous and subpopulations of TGF-&#946;/Smad active cells are responsible for biological function in a time-dependent manner8,9. Common cellular analysis of TGF-&#946;/Smad signaling involves the use of fixed endpoint assays that provide only a snapshot of cellular activity and often quantitate the average TGF-&#946;/Smad effect10. These methods, however, may not accurately represent the molecular behaviors of TGF-&#946;/Smad signaling in the physiological state during disease progression. Image-based analysis of live cells capture the dynamics of cellular and biological processes with both a spatial and temporal understanding.
Our goal was to develop a sensitive high-throughput method for live cell imaging of TGF-&#946;/Smad signaling using adenovirus-based reagents. Here, we infected the human breast cancer cell line MDA-MB-231 with an adenovirus expressing the Smad3 CAGA motif binding sequence and a luciferase (Luc) or Td-Tomato (Td-Tom) reporter gene. Adenoviral reporter systems provide a quick and cheap method for plasmid introduction that can result in a 100% infection rate in cancer cell lines. Adenoviral reporter systems have also been successfully applied to cell lines that are difficult to transfect with conventional plasmid11. In this protocol we will describe a reproducible and noninvasive process to achieve live cell imaging of the TGF&#946;/Smad signaling pathway both in vivo and in vitro.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>1881024</td>
			<td>Warm in 37 &#176;C waterbath before use</td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Scientifix Life</td>
			<td>FBS500-S</td>
			<td>Heat inactivated before use</td>
		</tr>
		<tr>
			<td>Recombinant Human TGF-&#946;1</td>
			<td>PEPROTECH</td>
			<td>100-21</td>
			<td>Aliquot in DDW to make final concentration at 10 mg/mL</td>
		</tr>
		<tr>
			<td>Hoechst-33258</td>
			<td>Tocris Bioscience</td>
			<td>5117</td>
			<td>Dilute in to PBS to make final concentration at 1 &#956;L/mL</td>
		</tr>
		<tr>
			<td>Luciferase Reporter Assay Kit</td>
			<td>Promega</td>
			<td>197897</td>
			<td>Dilute 5x in PBS before use</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>9100-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast fluorescence microscopy</td>
			<td>OLYMPUS</td>
			<td>IX50</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>VivoGl Luciferin</td>
			<td>Promega</td>
			<td>P1041</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina III In Vivo Imaging System</td>
			<td>PerkinElmer</td>
			<td>CLS136334</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5% Trypsin-EDTA (10x)</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td>Diltue to 0.05% (1x) in PBS</td>
		</tr>
		<tr>
			<td>Cell Culture Lysis 5x Reagent</td>
			<td>Promega</td>
			<td>E153A</td>
			<td>Dilute to 1x in DDW</td>
		</tr>
		<tr>
			<td>10% Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>F5554-4L</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293A</td>
			<td>ThermoFisher</td>
			<td>R70507</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA-MB-231</td>
			<td>ATCC</td>
			<td>CRM-HTB-26</td>
			<td></td>
		</tr>
		<tr>
			<td>PRKDC-SCID</td>
			<td>Animal Resources Centre</td>
			<td>SCIDF6</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Chem-supply</td>
			<td>EA043-10L-P</td>
			<td></td>
		</tr>
		<tr>
			<td>Refresh Night Time</td>
			<td>Allergan</td>
			<td>1750D</td>
			<td>Lubricating Eye Ointment</td>
		</tr>
		<tr>
			<td>Solution</td>
			<td></td>
			<td></td>
			<td>Composition</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td>NaH2PO4.2H2O (4 mM); NaHPO4 (16 mM); NaCl (0.12M)</td>
		</tr>
		<tr>
			<td>FBS-DMEM&#160;</td>
			<td></td>
			<td></td>
			<td>5% heat inactivated FBS; 10 &#956;g/mL penicillin; 100 &#956;g/mL streptomycin</td>
		</tr>
	</tbody>
</table>

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 single cell imaging in vitro
To accurately assess the activation of TGF-&#946;/Smad signaling in single cells using adenovirus based reagents, it is important to first determine the optimal Multiplicity of Infection (MOI) for each cell line. The optimal MOI is determined when 100% of cells are positive for adenoviral infection with no present cytopathic or cytotoxic effects. To determine this, we used a constitutively...

Watch this Scientific Journal Video about Live Cell Imaging of the TGF-	ÃŽÂ²/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System at JoVE.com

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo 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 ...

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Research

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Cancer Research

10.3K Views.  University of Melbourne. Here, we present a protocol for live cell imaging of TGF-β/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. 

Video: Live Cell Imaging of the TGF-	β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

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 Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

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

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

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

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

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.

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 ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

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 ...

In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by introducing specific mutations in oncogenes and tumor suppressor genes, using clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome editing technology in mice. This technology allows us to accurately mimic the genetic changes that occur in human cancers using mice. By genetically transforming murine primary cells, we can better study cancer development, progression, treatment, and diagnosis. In this study, we used Cre-inducible Cas9 mouse tongue epithelial cells to enable genome editing using adeno-associated virus (AAV) in vitro. Specifically, by altering KRAS, p53, and APC in normal tongue epithelial cells, we generated a murine head and neck cancer (HNC) cell line in vitro,which is tumorigenic in syngeneic mice. The method presented here describes in detail how to generate HNC cell lines with specific genomic alterations and explains their suitability for predicting tumor progression in syngeneic mice. We envision that this promising method will be informative and useful to study tumor biology and therapy of HNC.

Development of murine models with specific genes mutated in head and neck cancer patients is required for understanding of neoplasia. Here, we present a protocol for in vitro transformation of primary murine tongue cells using an adeno-associated virus-Cas9 system to generate murine HNC cell lines with specific genomic alterations.

The use of primary normal epithelial cells makes it possible to reproducibly induce genomic alterations required for cellular transformation by ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.