The Yeh lab is supported by research funding from an American Cancer Society Institutional Research Grant (IRG-97-219-14) awarded to the Hollings Cancer Center at MUSC, by research funding from a Department of Defense grant (W81XWH-11-2-0229) at MUSC, and by an award from the Concern Foundation (to ESY).

The protocol follows the guidelines and animal care standards of the Medical University of South Carolina (MUSC) and its Institutional Animal Care and Use Committee (IACUC).
1. Lung Dissection
<ol>
	<li>Mammary tumor dissection (optional depending on study goals and whether primary tumor analysis or tail vein injection was performed).
	<ol>
		<li>Euthanize mouse using a lethal dose of isoflurane anesthesia according to IACUC and university regulation. Confirm euthanization by surgical dislocation.&#160;</li>
		<li>On foam board, pin with dissection pins by placing mouse on its back with limbs spread.</li>
		<li>Spray mouse with 70% ethanol.</li>
		<li>Using forceps, grasp the lower portion of the animal at the center.</li>
		<li>Using scissor, cut upward toward the neck through the skin, being careful not to pierce the peritoneal cavity of the mouse.</li>
		<li>Cut back skin at the limbs to reveal tumor tissue.</li>
		<li>Dissect out tumor tissue and preserve by preferred method, such as freezing or fixation, for further analysis (not discussed further in this protocol).&#160;</li>
	</ol>
	</li>
	<li>Lung tissue dissection.
	<ol>
		<li>If tumor tissue was harvested as described above, pin down any excess skin.</li>
		<li>Using forceps, grasp peritoneum at lower end of the animal and begin cutting upwards toward the base of the neck.</li>
		<li>Carefully cut along the left and right sides of the rib cage being careful to avoid severing any blood vessels that may bleed into the thoracic cavity. Remove the rib bones by cutting to the left and right through the diaphragm and upper portions of the rib cage.</li>
		<li>Using forceps grasp the trachea of the mouse and pull forward.</li>
		<li>Sever the trachea with dissection scissors and remove lungs from mouse. 
		Note: The heart may remain attached to the lung. If this occurs, carefully dissect heart away from lungs being careful to collect all five lobes (see Figure 1).</li>
		<li>Wash gently with PBS to remove excess blood.</li>
	</ol>
	</li>
	<li>Gross evaluation of lung tissue.
	<ol>
		<li>Option 1: In vivo imaging.
		<ol>
			<li>For luciferase imaging, ~10 min prior to imaging, give animals a 150 mg/kg dose of luciferin by intraperitoneal (i.p.) injection. Image lungs in vivo in anesthetized animals or upon removal after dissection (Figure 2) 5.</li>
		</ol>
		</li>
		<li>Option 2: Gross evaluation.
		<ol>
			<li>Examine lungs under stereo microscope to visualize tumor nodules. Note: If cells are GFP labeled, a fluorescence capable stereomicroscope containing a light cube with the appropriate excitation/emission maxima (e.g. near 470/510 nm) to detect GFP may be used to examine lungs stereoscopically for fluorescence prior to processing.</li>
		</ol>
		</li>
		<li>If lungs are to be analyzed immediately, proceed to &#39;RNA Isolation&#39; section. Otherwise, snap freeze tissue using liquid nitrogen or dry ice. Store at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. RNA Isolation
Note: For the representative analysis, RNA was isolated using a RNA isolation kit (see Materials List). While the current analysis uses one specific manufacturer&#39;s product, a variety of isolation kits are available from several reputable vendors. Additionally, non-kit based centrifugation methods are also an option. cDNA should also be prepared from a positive control RNA sample, such as a cell line or plasmid, for standard curve analysis. Alternatively, a positive control specific for the primer probe set may be purchased (see Materials List).
<ol>
	<li>If using previously frozen tissue, gather frozen tissue on dry ice.</li>
	<li>Homogenize tissue using one of the following methods such as: mortar and pestle grinding performed by hand over dry ice after freezing tissue in liquid nitrogen; tissue homogenizer or mechanical dissociation device per manufacturer&#39;s suggestion; sonication, or other preferred method. 
	Note: For the representative analysis, the preferred method of homogenization is sonication.
	<ol>
		<li>For the analysis presented in Figure 3, prepare lysis buffer (provided in RNA isolation kit) and 2-mercaptoethanol at a ratio of 20 &#181;l 2-mercaptoethanol for every 1 ml of lysis buffer. Resuspend tissue in 300 &#181;l lysis buffer supplemented with 2-mercaptoethanol for every 30 g of tissue and sonicate for 10 sec with sonicator set at 30% amplitude.</li>
		<li>If using mortar and pestle, resuspend ground tissue in 300 &#181;l lysis buffer after grinding.</li>
	</ol>
	</li>
	<li>Add 10 &#181;l proteinase K to 590 &#181;l TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). Add 600 &#181;l proteinase K solution to every 300 &#181;l (i.e. for every 30 mg) of tissue. Incubate for 10 min at RT. 
	Note: Digestion time may vary.&#160;&#160;</li>
	<li>Centrifuge samples at &#8805;13,000 g for 10 min to remove debris.</li>
	<li>Transfer supernatant to new tubes.</li>
	<li>Add 450 &#181;l ethanol (96%-100%) for every 900 &#181;l of supernatant to precipitate RNA.</li>
	<li>Transfer 700 &#181;l of the lysate/ethanol mix to the column.</li>
	<li>Centrifuge samples at &#8805;13,000 g for 1 min to bind RNA to column.</li>
	<li>Discard liquid waste and add remaining supernatant to column and spin again.</li>
	<li>Once all the lysate is spun through the column, add 350 &#181;l of Wash Buffer 1 to the upper portion of the column and centrifuge samples at &#8805;13,000 g for 30 sec.</li>
	<li>Discard liquid from column and replace upper portion.</li>
	<li>Prepare DNase by mixing 5 units of DNase I enzyme with 5 &#181;l of 10x DNase and 40 &#181;l of DNase/RNase free water per sample (total volume of 50 &#181;l/sample).</li>
	<li>Add 50 &#181;l of DNase mix to each column and incubate for 15 min at RT.</li>
	<li>Add 350 &#181;l of Wash Buffer 1 to column and centrifuge samples at &#8805;13,000 g for 30 sec.</li>
	<li>Discard liquid from column and replace upper portion.</li>
	<li>Add 600 &#181;l of Wash Buffer 2 to the column and spin 30 sec at &#8805;13,000 g.</li>
	<li>Discard liquid from column and replace upper portion.</li>
	<li>Add 250 &#181;l of Wash Buffer 2 and centrifuge for 2 min at &#8805;13,000 g.</li>
	<li>Put part of column into new collection tube and discard old collection tube.</li>
	<li>Add 50-100 &#181;l of RNase/DNase free water to column and incubate for 1 min.</li>
	<li>Spin for 1 min at &#8805;13,000 g.</li>
	<li>Re-run collected eluate through column to increase RNA yield.</li>
	<li>For immediate use, keep samples on ice and proceed to quantitation. For use later, snap freeze on dry ice or liquid nitrogen. Store at -80 &#176;C freezer until needed.</li>
</ol>
3. First Strand Synthesis
Note: For the representative analysis, the reverse transcriptase (RT) reaction was performed a First Strand cDNA synthesis kit designed for QRT-PCR (see Materials List).
<ol>
	<li>If samples were previously collected, thaw tubes on ice.</li>
	<li>Measure RNA quantity using a spectrophotometer.</li>
	<li>Using 0.5-2 &#181;g of total RNA, perform first strand synthesis using reverse transcriptase to generate cDNA stock.
	<ol>
		<li>In sterile, RNase/DNase-free PCR tubes/plates, prepare the reaction mix (Table 1). Mix gently and centrifuge. Program a standard PCR machine (Table 2).</li>
	</ol>
	</li>
	<li>Dilute finished reaction to desired volume (generally 50-100 &#181;l) using sterile nuclease-free water.</li>
</ol>
4. Real-time PCR
Note: For the representative analysis, SYBR green was used. However, any preferred method can be substituted at the user&#39;s discretion.
<ol>
	<li>Using a positive control cDNA, set up a standard curve for each primer set.
	<ol>
		<li>For the analysis shown in Figure 3, prepare the standard curve for human HER2 using the manufacturer recommended positive control cDNA (see Materials List). For mouse gapdh, use the negative control lung cDNA to generate a standard curve. For each probe, serially dilute the cDNA 1:4 to generate 5 standards.</li>
	</ol>
	</li>
	<li>Calculate the number of total samples, which should include the standard curve and experimental samples. 
	Note: For the analysis described here, 2-3 experimental replicates per sample were analyzed. Standard curve analysis was performed to generate a simple linear regression model that can be applied to determine the relative amount of HER2 and gapdh per sample to calculate final normalized quantity. This analysis will also ensure that the primer efficiency is adequate for the analysis at hand.</li>
	<li>In a sterile, RNase/DNase-free tube, prepare master mix (Table 3).
	<ol>
		<li>Calculate master mix amount per sample. Scale up for more than one sample. Use a master mix for each experimental gene of interest, as well as an internal control such as gapdh. Use primers for gapdh at a final concentration of 200 nM. 
		Note: The internal control is particularly useful when trying to distinguish between human and mouse cell origin. 
		Note: Primer concentration for HER2 was optimized and determined by the manufacturer.</li>
	</ol>
	</li>
	<li>Prepare test plate containing 1 &#181;l cDNAs corresponding to each standard or experimental sample. Allocate at least 1 cDNA sample/well for each primer probe. Add 19 &#181;l of master mix per well for each primer probe. 
	Note: For the representative data in Figure 3, the cDNA samples were analyzed in duplicate for each primer probe and graphed as the average of the two samples.</li>
	<li>Run reaction in QRT-PCR machine using recommended or previously tested PCR protocol. See Table 4 for the PCR protocol used for the representative data in Figure 3 and Figure 4.&#160;</li>
</ol>

<ol>
	<li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+building+a+framework.">Cancer metastasis: building a framework.</a> Cell. 127 (4), 679-695 (2006).</li><li>Bos, P. D., Nguyen, D. X., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+metastasis+in+the+mouse.">Modeling metastasis in the mouse.</a> Curr Opin Pharmacol. 10 (5), 571-577 (2010).</li><li>Kim, I. S., Baek, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+breast+cancer+metastasis.">Mouse models for breast cancer metastasis.</a> Biochem Biophys Res Commun. 394 (3), 443-447 (2010).</li><li>Saxena, M., Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rebuilding+cancer+metastasis+in+the+mouse.">Rebuilding cancer metastasis in the mouse.</a> Mol Oncol. 7 (2), 283-296 (2013).</li><li>Yang, S., Zhang, J. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+tumor+metastasis.">Mouse models for tumor metastasis.</a> Methods Mol Biol. 928, 221-228 (2012).</li><li>Rampetsreiter, P., Casanova, E., Eferl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+mouse+models+of+cancer+invasion+and+metastasis.">Genetically modified mouse models of cancer invasion and metastasis.</a> Drug Discov Today Dis Models. 8 (2-3), 67-74 (2011).</li><li>Condeelis, J., Segall, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+cell+movement+in+tumours.">Intravital imaging of cell movement in tumours.</a> Nat Rev Cancer. 3 (12), 921-930 (2003).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17 (5), 545-580 (2003).</li><li>Zinn, K. R., 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=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49 (1), 103-115 (2008).</li><li>Tseng, J. C., Kung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+bioluminescence+imaging+of+mouse+tumor+models.">Quantitative bioluminescence imaging of mouse tumor models.</a> Cold Spring Harb Protoc. 2015 (1), (2015).</li><li>Radonic, 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=Guideline+to+reference+gene+selection+for+quantitative+real-time+PCR.">Guideline to reference gene selection for quantitative real-time PCR.</a> Biochem Biophys Res Commun. 313 (4), 856-862 (2004).</li></ol>

ESY and MAA are not employed by FirstString Research Inc. and do not hold any financial interest in the company. FirstString Research Inc. provided the luciferase imaging data that was presented in this manuscript. GSG is president and CEO of FirstString Research. CLG is an employee of FirstString Research. GSG and CLG have stock options issued by FirstString Research.

This protocol describes use of QRT-PCR to evaluate mammary tumor cell metastasis to the lung using a xenograft mouse model. It is acknowledged that other techniques, including bioluminescent imaging, are available and are also effective for detecting metastasis despite having their own values and limitations. The data presented suggests that QRT-PCR provides an effective measure of detecting tumor-derived mRNA within lung tissue for quantitation of total metastasis. It is proposed that QRT-PCR analysis provides a seconda...

QRT-PCR analysis is proposed as a method for assessing tumor metastasis. This method is proposed as an alternative for users that are interested in evaluating metastasis but may not have access to specific equipment such as in vivo imaging equipment or a fluorescence capable stereoscope. A discussion of commonly used methods is presented followed by a demonstration for how QRT-PCR analysis can be used either as a separate or as a companion method to evaluate metastasis. This procedure has the potential to provide a quantitative analysis of metastatic burden.
Standard methods of gross analysis, including visualization of lungs under a stereomicroscope as well as serial sectioning followed by hematoxylin and eosin (H&#38;E) staining of lung tissue, are quantifiable but rely heavily on user defined parameters for counting 2-5. When evaluating whole lungs using a stereomicroscope, only large surface metastases are visible and analysis requires the investigator to have reasonable knowledge of lung anatomical structure to determine what constitutes a metastatic lesion. Fluorescent labeling of tumor cells with a marker such as GFP and use of a stereomicroscope that contains a light cube with the appropriate excitation/emission maxima (e.g. near 470/510 nm for GFP) assists in this process, but only surface tumor nodules are detectable. Additionally, fluorescence from blood contamination, which is visible under the same parameters as GFP, may lead to false identification of possible metastatic lesions.
Sectioning of the lung followed by H&#38;E staining to visualize lung metastasis is a useful method to evaluate micrometastases and other microscopic processes including immune cell infiltration but often requires use of the entire lung tissue for paraffin embedding, sectioning, and staining procedures. Therefore, downstream procedures are not ideal following this method. Although quantifiable, this procedure requires the investigator to evaluate a large number of stained lung sections per animal to ensure that the analysis accounts for the entire 3D structure of the lung. Consequently, this type of examination is time consuming, can lead to counting error, and analysis relies heavily on investigator discretion.
Several in vivo imaging techniques (e.g. MRI, PET, SPECT) are currently used to perform or test biological processes in experimental rodent models 8. In vivo bioluminescent imaging is a common method used to acquire a gross view of metastasis 9. This technique is generally applied to evaluate the presence of luciferase reporter activity due to the accumulation of tumor cells, which are engineered to contain a luciferase response element, that reside in specific organs like the mammary gland after tumor cell implantation and the lung upon spontaneous metastasis 10. Visualization of luciferase reporter activity is induced by the presence of luciferin substrate (D-luciferin). Luciferase catalyzes the oxidative decarboxylation of D-luciferin to oxyluciferin generating bioluminescence. While informative, this method is limited by several factors including substrate stability (i.e. short half-life), adequate distribution of substrate which depends on how it is delivered to experimental animals, and low sensitivity of detection 9. A main merit to this technique is that it is non-invasive, can be performed on live animals, and can lead to the detection of tumor cell metastases in multiple organs that may not have been normally harvested at dissection 9,10.
One positive aspect of in vivo imaging techniques is that the lung tissue is undisturbed allowing for secondary procedures like paraffin embedding or as presented here, QRT-PCR analysis. However, because QRT-PCR is theoretically a more sensitive measure of detection, gross evaluation may not reveal low numbers of tumor cells present in the lung. While useful, the imaging techniques described above can be substituted or supplemented with the QRT-PCR method currently described. QRT-PCR has the potential to provide a sensitive measure of tumor-derived mRNA within a lung.

<table><tbody><tr><td>2-mercaptoethanol</td><td>Fisher</td><td>BP176-100</td><td></td></tr><tr><td>DNAse</td><td>Thermo Scientific</td><td>EN0521</td><td></td></tr><tr><td>GeneJet RNA Isolation Kit</td><td>Thermo Scientific</td><td>K0732</td><td></td></tr><tr><td>iScript Reverse Transcription Supermix for QRT-PCR</td><td>Bio-Rad</td><td>1708841</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix&amp;nbsp;</td><td>Bio-Rad</td><td>172-5121&amp;nbsp;</td><td></td></tr><tr><td>PrimePCR SYBR Green Assay: ERBB2, Human&amp;nbsp;</td><td>Bio-Rad</td><td>100-25636&amp;nbsp;</td><td></td></tr><tr><td>PrimePCR Template for SYBR Green Assay: ERBB2, Human&amp;nbsp;</td><td>Bio-Rad</td><td>100-25716&amp;nbsp;</td><td></td></tr><tr><td>gapdh Primer Sequence</td><td></td><td></td><td>For-ATGGTGAAGGTCGGTGTGAACG Rev-CGCTCCTGGAAGATGGTGATGG</td></tr></tbody></table>

evaluation of lung metastasis in mouse mammary tumor models by quantitative real-time pcr

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

Beyond the time it takes to perform the initial inoculation of tumor cells into the experimental animal and if performing, primary in vivo tumor analysis, the tissue harvest, RNA isolation, and QRT-PCR analysis is a 1-2 day procedure (Figure 1).
An example of gross analysis is bioluminescent imaging to evaluate tumor cells within the lung tissue. Here, a mammary tumorigenesis experiment was performed to...

Watch this Scientific Journal Video about Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR at JoVE.com

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

evaluation-lung-metastasis-mouse-mammary-tumor-models-quantitative

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9.8K Views.  Medical University of South Carolina. This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Video: Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

Cancer Research

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

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.

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 ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

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