Name: combined use of tail vein metastasis assays and real-time in vivo imaging to quantify breast cancer metastatic colonization and burden in the lungs
Uploaded: 2019-12-19T16:00:00
Description: Watch this Scientific Journal Video about Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs at JoVE.co

We thank Emily Norton for assisting with viral infections and critical reading of the manuscript. We also thank Ryan Kanai for help with acquiring images of the lungs and Kate E. Tubbesing for help with image analysis of the green metastases in the lungs. We thank the animal research facility staff for their support and for assistance in the preparation of this video. This work was supported by a Susan G. Komen Career Catalyst Grant that awarded to J.M.L. (#CCR17477184).

This protocol involves the use of mice and biohazardous materials and requires approval from the appropriate institutional safety committees. All of the described in vivo work here is approved by the Albany Medical College institutional animal care and use committee (IACUC).
NOTE: For a protocol overview, see schematic in Figure 1.
1. Packaging all required retroviruses and lentiviruses
NOTE: The described prot...

Critical steps of the method 
It is critical to optimize the number of cells injected (step 3) for a given cell line and mouse strain as this can greatly influence the number of metastases that form and the length of the experiment. If too many cells are injected or the metastases grow for too long, the metastases may be difficult to count making the effects of the genetic manipulation difficult to assess. However, if too few cells are injected, few or no metastases may form. Thus, a preliminary exp...

Cancer remains the second leading cause of death worldwide1 and metastasis is responsible for the majority of these deaths2,3. However, a limited understanding of the molecular mechanisms that govern metastatic colonization and subsequent growth has hindered the development of effective treatments for metastatic disease. The identification of novel therapeutic targets requires an assay to test how perturbed expression or function of a candidate gene influences metastasis formation and growth. While autochthonous mouse models have their advantages, they are time-consuming and expensive to generate, making them more suited for target validation rather than target discovery. Transplant model systems in which the candidate gene is perturbed in cancer cells in vitro and then effects on metastatic potential are assessed in vivo, are less expensive and higher throughput than autochthonous models. In addition, viral vectors for stable delivery of RNAi, CRISPR/CAS9, and transgenes are widely available, making it relatively easy to perturb virtually any gene or genes of interest in a cancer cell lines. This approach can also be used to assay the role of candidate genes in metastatic colonization and growth in human cancer cell lines by transplanting the cells into immunocompromised or humanized mice.
The two types of assays used to test metastasis formation by transplanted cancer cells in vivo are spontaneous metastasis assays and experimental metastasis assays. In spontaneous metastasis assays4,5, cancer cells are injected into mice, allowed to form a primary tumor, and then spontaneous metastasis formation and subsequent growth are assayed. The strength of this model is that the cells must complete all steps of the metastatic process in order to form metastatic tumors. However, many cancer cell lines do not metastasize efficiently in spontaneous metastasis models, and any manipulation of the cells that impacts primary tumor growth can confound the results of the metastasis assay. Experimental metastasis assays, in which cancer cells are injected directly into circulation, are used to avoid these pitfalls. Common experimental metastasis assays include the tail vein injection6,7,8 (and demonstrated here), intracardiac injection9, and portal vein injection10.
The purpose of the protocol presented here is to provide an in vivo experimental metastasis assay that allows a researcher to monitor metastasis formation and growth in real time, as well as to quantify end point metastasis number and size in the lungs of the same mouse. To accomplish this, traditional experimental tail vein metastasis assays6,7,8 are combined with live animal imaging, using an in vivo imaging device9,11,12,13,14. Tumor cells stably expressing both luciferase and a fluorescent protein are injected into mice via the lateral tail vein and then the in vivo imaging device is used to measure changes in metastatic burden in the lungs over time (Figure 1). However, the in vivo live animal imaging device cannot distinguish or measure the size of individual metastases. Thus, at the end of the experiment, a fluorescent stereomicroscope is used to count the number and measure the size of the fluorescent metastases in the lungs without the need for sectioning and histology or immunohistochemistry (Figure 1). This protocol can be used to test how altering the expression or function of a candidate gene influences metastasis formation and growth. Potential therapeutic compounds such as small molecules or function blocking antibodies can also be tested.
To demonstrate this approach, we first performed a proof of concept experiment in which the essential replication factor, replication protein A3 (RPA3) is knocked down in metastatic mouse breast cancer cells. We show that mice injected with RPA3 knockdown cells have significantly less metastatic burden at every time point compared to mice injected with control cells. Analysis of the metastasis-containing lungs shows that this reduced metastatic burden is the result of significantly reduced metastatic colonization and impaired growth of the metastases that form. To further demonstrate this technique, we tested whether simultaneous knock down of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) impairs metastatic colonization or subsequent growth. YAP and TAZ are two related transcriptional co-activators that are the critical downstream effectors of the Hippo Pathway. We15,16 and others have implicated YAP and TAZ in metastasis (reviewed in17,18,19), suggesting that these proteins are good therapeutic targets. Consistently, we found that mice injected with YAP/TAZ knockdown cells had significantly reduced metastatic burden. Analysis of the lungs showed that the YAP/TAZ knockdown cells formed many fewer metastases and that the metastases that did form were smaller. These experiments demonstrate how experimental metastasis assays allow a researcher to quickly and inexpensively test the role of a candidate gene in metastasis formation and growth. They further show how the combined use of live animal imaging and fluorescent quantification of metastases in whole lungs allows the researcher to better understand the steps during metastatic colonization.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% SDS-PAGE Gel</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>2.5% Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>Trypsin for tissue culture</td>
		</tr>
		<tr>
			<td>96 well flat bottom white assay plate</td>
			<td>Corning</td>
			<td>3922</td>
			<td>For measuring luciferase and renilla signal in cultured cells</td>
		</tr>
		<tr>
			<td>Alcohol wipes</td>
			<td></td>
			<td></td>
			<td>For sterolizing the injection site before tail vein injecitons</td>
		</tr>
		<tr>
			<td>BALB/C mice (female, 6 weeks)</td>
			<td>Taconic</td>
			<td>BALB-F</td>
			<td>For tail vein metastatic colonization and burden assays</td>
		</tr>
		<tr>
			<td>BSA regular</td>
			<td>VWR Ameresco</td>
			<td>97061-416</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Cell lysis buffer</td>
			<td>Cell Signaling</td>
			<td></td>
			<td>For collecting protien samples</td>
		</tr>
		<tr>
			<td>Celltreat Syringe Filters, PES 30mm, 0.45 &#956;m</td>
			<td>Celltreat</td>
			<td>40-229749-CS</td>
			<td>For filtering viral supernatant</td>
		</tr>
		<tr>
			<td>CO2 and euthanasia chamber</td>
			<td></td>
			<td></td>
			<td>For euthanasing the mice</td>
		</tr>
		<tr>
			<td>Dual-luciferase reporter assay kit</td>
			<td>Promega</td>
			<td>E1960</td>
			<td>For measuring luciferase and renilla signal in cultured cells</td>
		</tr>
		<tr>
			<td>Dulbecco&39;s phosphate buffered saline</td>
			<td>Himedia</td>
			<td>TS1006</td>
			<td>For PBS</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>VWR</td>
			<td>97061-406</td>
			<td>Used to dilute trypsin for tissue culture</td>
		</tr>
		<tr>
			<td>FBS 100% US origin</td>
			<td>VWR</td>
			<td>97068-085</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Fujifilm LAS-3000 gel imager</td>
			<td>Fujifilm</td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>GAPDH(14C10) Rabibit mAb</td>
			<td>Cell Signaling</td>
			<td>2118</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) Secondary Antibody, HRP conjugate</td>
			<td>Thermo Scientific</td>
			<td>31460</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Human embryonic kidney cells, HEK-293FT</td>
			<td>Invitrogen</td>
			<td>R70007</td>
			<td>Cell line used for packging virus</td>
		</tr>
		<tr>
			<td>HyClone DMEM/High clucose</td>
			<td>GE Healthcare life sciences</td>
			<td>SH30243.01</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Hygromycin B, Ultra Pure Grade</td>
			<td>VWR Ameresco</td>
			<td>97064-810</td>
			<td>For antibiotic selection of infected cells</td>
		</tr>
		<tr>
			<td>I3-P/i3 Multi-Mode Microplate/EA</td>
			<td>Molecular devices</td>
			<td></td>
			<td>For measuring luciferase and renilla signal in cultured cells</td>
		</tr>
		<tr>
			<td>Imagej</td>
			<td></td>
			<td></td>
			<td>Used for image analysis of lung metastases: threshold set to 25 &#38; 100</td>
		</tr>
		<tr>
			<td>Immuno-Blot PVDF Membrane</td>
			<td>Biorad</td>
			<td>1620177</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td></td>
			<td></td>
			<td>For mouse anesthesia</td>
		</tr>
		<tr>
			<td>IVIS Lumina XRMS In Vivo Imaging System (in vivo live animal imaging device)</td>
			<td>PerkinElmer</td>
			<td>CLS136340</td>
			<td>For in vivo imaging of metastatic burden</td>
		</tr>
		<tr>
			<td>Leica M205 FA &#38; Lecia DCF3000 G (GFP and bright field filters)</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Microscope and camera for visualing, counting and taking pcitures of metastases in the lungs; 10X magnifacation, 3.5 sec exposure, 1.4 gain</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000</td>
			<td>Life technologies</td>
			<td>L3000008</td>
			<td>For YAP/TAZ-TEAD reporter transfection</td>
		</tr>
		<tr>
			<td>Living Image 3.2 (image software program)</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>Software for IVIS</td>
		</tr>
		<tr>
			<td>Mouse breast cancer cells, 4T1</td>
			<td>Karmanos Cancer Institute</td>
			<td>Aslakson, CJ et al.,1992</td>
			<td>Mouse metastatic breast cance cell line</td>
		</tr>
		<tr>
			<td>Multi-Gauge version 3.0</td>
			<td>Fujifilm</td>
			<td></td>
			<td>Software for quantifying western blot band intensity</td>
		</tr>
		<tr>
			<td>Opti-MEM (transfection buffer)</td>
			<td>Gibco</td>
			<td>31985-062</td>
			<td>For packaging virus and transfection</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Component of complete growth media</td>
		</tr>
		<tr>
			<td>Pierce BCA protein assay kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>For quantifying protein concentration</td>
		</tr>
		<tr>
			<td>Pierce Phosphatase Inhibitor Mini Tablets</td>
			<td>Thermo Scientific</td>
			<td>A32957</td>
			<td>Added to cell lysis buffer</td>
		</tr>
		<tr>
			<td>Pierce Protease Inhibitor Mini Tablets</td>
			<td>Thermo Scientific</td>
			<td>A32953</td>
			<td>Added to cell lysis buffer</td>
		</tr>
		<tr>
			<td>Polybrene (hexadimethrine bromide)</td>
			<td>Sigma-Aldrich</td>
			<td>45-H9268</td>
			<td>For infection</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Sigma-Aldrich</td>
			<td>45-P7255</td>
			<td>For antibiotic selection of infected cells</td>
		</tr>
		<tr>
			<td>Rodent restrainer</td>
			<td></td>
			<td></td>
			<td>For restraining mice during tail vein injeciton</td>
		</tr>
		<tr>
			<td>SDS-PAGE running buffer</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>TAZ (V3886) Antibody</td>
			<td>Cell Signaling</td>
			<td>4883</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>TBST buffer</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>TC20 automated cell counter</td>
			<td>Bio-Rad</td>
			<td></td>
			<td>For counting cells</td>
		</tr>
		<tr>
			<td>Vectors</td>
			<td></td>
			<td></td>
			<td>See Table 1 for complete list of vectors</td>
		</tr>
		<tr>
			<td>VWR Inverted Fluorescence Microscope</td>
			<td>VWR</td>
			<td>89404-464</td>
			<td>For visualizing fluorescence in ZSGreen labeled cells</td>
		</tr>
		<tr>
			<td>Western transfer buffer</td>
			<td></td>
			<td></td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>XenoLight D-Luciferin K+ Salt</td>
			<td>PerkinElmer</td>
			<td>122799</td>
			<td>Substrate injected into mice for in vivo bioluminescent IVIS images</td>
		</tr>
		<tr>
			<td>X-tremeGENE 9 DNA transfection reagent (lipid solution for transfection)</td>
			<td>Roche</td>
			<td>6365787001</td>
			<td>For packaging virus</td>
		</tr>
		<tr>
			<td>YAP (D8H1X) XP Rabbit mAb</td>
			<td>Cell Signaling</td>
			<td>14074</td>
			<td>For western blot</td>
		</tr>
	</tbody>
</table>

combined use of tail vein metastasis assays and real-time in vivo imaging to quantify breast cancer metastatic colonization and burden in the lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

To demonstrate the above approach, we performed a proof of concept experiment in which a critical replication factor, RPA3 was knocked down in a metastatic mouse mammary carcinoma cell line (4T122). While the protocol describes labeling the cells with both luciferase and fluorescent proteins prior to genetic manipulation, we used a modified approach because THE RNAi vectors also deliver ZsGreen (Figure 2A). First, 4T1 cells were stably transduced with...

Watch this Scientific Journal Video about Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs at JoVE.co

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Our protocol can be used to quickly and easily test the role of any candidate gene in metastatic colonization and growth. The protocol also allows realtime monitoring of metastasis, formation, and growth, as well as quantification of metastasis number and size in the same animals without the need for resectioning or histological staining. Plate four T1 cells at 150, 000 cells per well in a 12-well plate in complete growth media.Incubate at 37 degrees Celsius 5%carbon dioxide overnight. The next day, aspirate the growth media from the cells. Add 500 microliters of luciferase viral supernatant and 500 microliters of fluorescent protein viral supernatant to simultaneously infect the cells with both the luciferase and fluorescent protein viral supernatants.Then, add one microliter of eight milligram per milliliter hexadimethrine bromide and incubate at 37 degrees Celsius and 5%carbon dioxide for 24 to 48 hours. Trypsinize the four T1 cells with 500 microliters of trypsin. After two to five minutes, transfer all the cells to a six centimeter dish in four milliliters of selection media, thereby quenching the trypsin.After selection is complete, confirm that the infected four T1 cells are expressing ZsGreen fluorescent protein using an inverted fluorescent microscope at 50 to 100 times magnification. Also confirm that the infected four T1 cells are expressing luciferase using a commercially available luciferase activity kit. Proceed to perform optimization of the in vivo experimental design as described in the text protocol.Aspirate the media and rinse the cell plates with 1X PBS. Trypsinize the cells with five milliliters of trypsin per 15 centimeter plate for two to five minutes and transfer all of the cells to a conical tube. Wash remaining cells from the tissue culture dish with enough complete growth media to quench the trypsin.Add the wash to the same conical tube. Count the cells using an automated cell counter to determine the total cell number. Next, centrifuge the cells at 122 times G for three minutes, and aspirate the supernatant.Re-suspend the cells in 1X PBS at the desired concentration. Here, 25, 000 cells are injected into each mouse and 100 microliters of PBS. So the re-suspended cells are at 250, 000 cells per milliliter.Keep the cell suspensions on ice until injection. Working in a hood at the animal facility, gently but thoroughly mix the cells by inverting the tube or using a one milliliter syringe to ensure that they are uniformly re-suspended. Now, load a one milliliter Luer Lock syringe with the cell suspension and expel excess air bubbles.Place a half inch 30 gauge needle on the syringe with the bevel up and expel air bubbles. Gently place the mouse in a rodent restrainer. The lateral tail vein should be visible and dilated.If not, gently pinch the base of the tail and dip the tail in warm tap water to dilate the veins. Use an alcohol wipe to clean the tail, then insert the needle into the tail vein, bevel side up, and inject 100 microliters of cell suspension. If the needle is inserted properly into the vein, it should easily slide slightly forward and back, and there should not be resistance when the plunger is pushed.Successful injections should also result in a flush in which the blue color of the vein turns white for a few seconds following the injection. Slowly remove the needle, and using a sterile gauze, apply pressure to the injection site to stop any bleeding. Return the mouse to its cage and monitor for 15 minutes to ensure full recovery.Turn on the in vivo live animal imaging device, open the image software, and log in. Click the initialize button and wait for the machine to initialize. Change the field of view to D.For first time use, edit exposure settings by clicking edit, preferences, acquisition, and auto exposure.Change the maximum exposure time from the default 60 seconds to 300 seconds and click okay. Load a one milliliter syringe with D-luciferin, then add a half inch 30 gauge needle to the syringe and expel air bubbles. Measure and record the mass of an anesthetized mouse.Restrain the mouse by pinching the scruff of the neck using the thumb and pointer fingers and grasping the tail between the pinky finger and the base of the hand. Invert the mouse at a 45 degree angle with its head pointing downward. Insert the needle, bevel side up, into the mouse's left side IP space.Confirm entry into the IP space by drawing back a small volume. There should not be color at the base of the needle when drawing back in the IP space. Inject the appropriate volume of D-luciferin for a dose of 150 milligrams per kilogram.Immediately after D-luciferin administration, start a timer. Place the mouse flat on its back in the imaging device with its nose in the nose cone, and ensure that one half to two and a half percent isoflurane is being administered. Click the luminescent and photograph boxes.Change the exposure time to auto, and then click acquire in the acquisition control panel. After imaging, return the mouse to its cage and monitor for 15 minutes to ensure full recovery. Following euthanasia, as described in the text protocol, isolate and remove the lungs from each mouse and rinse in 1X PBS to remove excess blood.Acquire images of ZsGreen metastases in the lobes at 10X in Brightfield and fluorescence using a fluorescent stereoscope with a GFP wide band filter. Use image analysis software to quantify the size and number of metastases from the images. To process and analyze the data from the images acquired with the in vivo live animal imaging device, open all image files for each mouse in the image software.Ensure that the units are in radiance for bioluminescent data and efficiency for fluorescent data by clicking the arrow at the top left of the image window and changing it to the appropriate unit. Use the image from the last time point to create a region of interest, or ROI, by first clicking ROI tools in the tool palette window. Then, insert one ROI by clicking the arrow and selecting one.Click the border of the ROI and move it over to the chest of the mouse. Adjust the size of the ROI so it covers the chest of the mouse and does not exclude signal. Then, click measure ROIs and copy or type the raw number into an Excel sheet.Plot and analyze the data as described in the text protocol. Right click in the image file to copy the ROI, then paste it into image files from the other time points and click measure ROIs. Retroviral vector expressing tandem miR-30-based shRNAs targeting both YAP and TAZ reduces YAP and tax protein expression and transcriptional activity in four T1 cells.Four T1 luciferase cells were stably transduced with a ZsGreen expressing version of this tandem YAP/TAZ shRNA vector or a controlled miR-30-based shRNA. Bioluminescent images show that the rate at which metastatic burden increased was significantly faster in the mice injected with control cells compared to the mice injected with YAP/TAZ knockdown cells. A plot of the log10 transformed luciferase signal measured for each mouse over the course of the experiment also shows that the rate was faster in the mice injected with the control cells compared to the mice injected with YAP/TAZ knockdown cells.Significantly fewer metastases formed in the mice injected with the YAP/TAZ knockdown cells compared to mice with control cells when counted manually. The same trend was observed when using the image analysis software. Not only did many more metastases form in the control mice, but they were generally larger.Consistently, the metastatic burden in the lungs was also drastically reduced by YAP/TAZ knockdown. Successful injection of equal numbers of healthy cells into each mouse is critical to ensure high quality data. Remember to use caution and follow safety guidelines when working with infectious retroviruses and lentiviruses, particularly if the viruses are human infectious.This technique has allowed us to illustrate the roles of candidate genes and metastatic colonization and growth, providing a way to identify and test novel therapeutic targets for the treatment of metastatic disease. Following this procedure, metastases containing lungs can be further analyzed by immunofluorescent or histological staining to investigate how the altered gene is influencing metastases and identify other proteins that may be involved.

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