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 fo:keep-together.within-page="1">
	<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&#160;</td>
			<td>Bio-Rad</td>
			<td>172-5121&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimePCR SYBR Green Assay: ERBB2, Human&#160;</td>
			<td>Bio-Rad</td>
			<td>100-25636&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimePCR Template for SYBR Green Assay: ERBB2, Human&#160;</td>
			<td>Bio-Rad</td>
			<td>100-25716&#160;</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

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

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 ...

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

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Models of Bone Metastasis

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

A Simple Method of Mouse Lung Intubation

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis