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 ...

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