We want to recognize Tyson Foods Inc. for generously providing the fertilized eggs. Shanna Arnold was instrumental during the filming of this protocol. 
Trenis Palmer was supported by the National Institutes of 
Health under the Ruth L. Kirschstein National Research Service Award (F31 National Cancer Institute). This work was partially supported by CCSRI Grant #700537 to JDL 
and NIH/NCI grant #CA120711-01A1 and CA120711-01A1 to AZ.

1. Preparing the eggs for xenografting (spontaneous metastasis)
<ol>
<li>Freshly laid fertilized chicken eggs are incubated in a rotating incubator for 10 days at 100&deg;F and 60% humidity. The eggs are rotated four times each hour.</li>
<li>On developmental day 10 the eggs are placed on their side in an egg rack. A goose-neck lamp or other suitable light source is used to candle the eggs by shining the light into the eggshell at the blunt end of the egg where the airsac is located.</li>
<li>The chorioallantoic vein is located and marked. This vein is located at the top of the eggshell where it is the junction of several large blood vessels. At this branch point the blood vessel drops down, away from the CAM and attaches to the embryo. A 1cm square box is drawn with pencil on the eggshell approximately 1cm away from the branch point in the vein. The area including and around the square is then cleaned using a cotton swab soaked in iodine.</li>
<li>Using a rotating cutting tool (Dremel) fitted with a silicone carbide grinding stone (Dremel part #84922) a hole is drilled through the blunt end of the egg into the air sac. Using this same tool a hole is drilled within the previously drawn square on top of the egg, stopping short of the eggshell membrane. A 25-gauge syringe needle with a fine bur on the end is used to make a small hole in the eggshell membrane being careful not to tear the underlying CAM. The CAM is subsequently detached from the eggshell membrane in the area of the square using gentle suction created with an automatic pipette aid fitted with a piece of &frac14;&#x201D; tygon tubing placed against the hole in the airsac. Upon applying suction, an air pocket should underneath the hole in the square signifying that the CAM has been successfully dropped away from the eggshell. After the CAM is dropped, the hole in the air sac and the hole located in the square near the chorioallantoic vein is sealed with a piece of tape laboratory tape. The eggs are subsequently placed into a stationary incubator at 100&deg;F and 60% humidity in anticipation of grafting the tumor cells. See figure 1 for a sequential depiction.</li>
</ol>
2. Preparing the eggs for intravenous injection (experimental metastasis)
<ol>
<li>Freshly laid fertilized chicken eggs are incubated in a rotating incubator for 12 days at 100&deg;F and 60% humidity. The eggs are rotated four times each hour.</li>
<li>On developmental day 12 the eggs are placed on their side in an egg rack. A goose-neck lamp or other suitable light source is used to candle the eggs by shining the light into the eggshell at the blunt end of the egg where the airsac is located.</li>
<li>The chorioallantoic vein is located and marked. A 1cm by 0.5 cm rectangle is drawn with pencil on the eggshell directly above the vein. The area including and around the square is then cleaned using a cotton swab soaked in iodine.</li>
<li>A small window is created in the eggshell in the drawn location using the Dremmel drill bit (#118). The window is removed to expose the underlying eggshell membrane which is subsequently rendered transparent with drop of mineral oil. See figure 5A for a graphical depiction.</li>
<li>At this point the hole in the air sac and the hole located in the square near the chorioallantoic vein is sealed with a piece of tape laboratory tape. The eggs are subsequently placed into a stationary incubator at 100&deg;F and 60% humidity in anticipation of injecting the tumor cells.</li> 
</ol>
3. Preparing tumor cells for grafting or injection.
<ol>
<li>For xenografting, the tumor cells are detached from from their culture dishes using Trypsin/EDTA and washed with 10 volumes of phosphate buffered saline (PBS) in order to remove any residual media, Trypsin, or EDTA.</li>
<li>For I.V. injection, cultured cells are detached using nonenzymatic cell-dissociation solution, and washed with serum-free DMEM. and)</li> 
<li>Cells are counted with a hemacytometer and resuspended in PBS at 10-40 million cells/ml for grafting, or alternatively resuspended in serum-free DMEM at 1 million cells/ml for I.V. injection.</li>
</ol>
4. Grafting the tumor cells onto the newly exposed CAM (Spontaneous Metastasis)
<ol>
<li>The eggs are removed from the incubator and a small window is cut in the location of the previously drawn square where the new air pocket has formed. This is most easily done with a rotary tool equipped with a cutting wheel (Dremel tool with the #409 circular cutting wheel). A pair of needle-nose forceps is used to remove the small section of eggshell and expose the underlying CAM.</li>
<li>Using a cotton-tipped applicator (Fisher Brand #23-400-001) the CAM is abraded 5 times. A little blood should be evident on the cotton. Immediately after damaging the CAM, 25&mu;l of the cell suspension is placed onto the damaged area. The optimal number of cells is determined empirically but can range from 0.5X105 to 1X106.The window in the egg is sealed tightly with tape and the chicks are left standing for 10-15 minutes in order to allow the cells to settle. After 15 minutes the eggs are returned to the stationary incubator.</li>
<li>The tumors are allowed to grow for 5-8 days depending on the specific application and the nature of the tumor cell line used. See figure 1 for a sequential depiction.</li>
</ol>
5. Intravenous injection of tumor cells (Experimental Metastasis)
<ol>
<li>Using a 30 Gauge insulin syringe 100&mu;l of cell suspension is injected into the allantoic vein of each embryo, the eggs were returned to a stationary incubator and remained there for an additional 1&#x2013;7 days.</li> 
<li>At distinct time points, the lung or lower CAM are harvested from each embryo and processed for the detection of tumor cells, as described below.</li>
</ol>
6. Harvesting tumors and chick tissues
<ol>
<li>A dissection area is prepared. The assay detects metastatic cells by quantifying human DNA using a highly sensitive PCR approach. It is therefore very important to prevent contamination with exogenous DNA. Dissections are performed in a dedicated area and throughout the dissections the tools are cleaned in between each animal. In order to prevent cross-contamination of your samples throughout the dissection process 3 separate sets of tools are used: one set for cutting the egg, one set for removing the primary tumor and one set for removing the internal organs. In addition, four wash containers are used for sequential rinsing of tools between each animal. In sequence these washes are: 1) double distilled water, 2) double distilled water, 3) chlorine bleach, and 4) 70% ethanol.</li>
<li>The eggs are removed from the stationary incubator and placed on ice for 15 minutes to anesthetize and euthanize the animals. The windows are opened such that the tumors become visible. Opening the windows bigger by removing some of the eggshell may be necessary. The primary tumors are resected from the CAM, weighed, and processed for histology.</li> 
<li>The chick is removed from the eggshell by cutting the shell radially into equal halves. The embryo is decanted from the cut shell and transferred to a clean weigh boat breast side up. The animal is dissected with using a fresh, clean set of tools. The animal is opened by cutting through the sternum. Once the embryo is open, a piece of the liver from either the right or left lobe is collected. The internal organs are subsequently removed by pulling gently on the esophagus attached to the gizzard and cutting the visera that connect the organs. In order to collect lungs cut the rib cage is opened the breast plate is separated and the heart is removed. At this point it is possible to cut around the four sides of the lung (the top closest to the head the left side along the rib cage, the bottom near the diaphragm and the right side along the trachea). For the sake of consistency, the same lung is collected in each animal. Harvested tissue samples are stored at -20&deg;C for later use or can be processed immediately for DNA isolation.</li>
</ol>
7. Genomic DNA isolation and quantitative PCR Analysis
<ol>
<li>Genomic DNA is extracted from tissues using the Sybr Green Extract&#x2013;N-Amp Tissue PCR Kit (Sigma-Aldrich Catalog #XNATRG). The extracted DNA can be transferred to a clean eppendorf tube and stored at -20&deg;C or used immediately. It is important to dilute the DNA 1:50 in nuclease free water before using it in the subsequent PCR reaction. Alternatively, DNA can be extracted using any number of standard DNA extraction protocols (Zijlstra et al., 2002).</li>
<li>Human DNA is detected in the chick tissues using quantitative PCR specific for human Alu sequences. Quantitative PCR using Alu-specific primers is performed using the SYBR green amplification from the Exract-N-Amp kit. The PCR kit comes with a 2x reaction mix containing SYBR green, buffer, salts dNTPS, Taq polymerase and the Jumpstart Taq Antibody. The reaction mix is made up in a final volume of 15&mu;l, with 0.4&mu;M of each primer. For most instances using the extracted DNA diluted 1:50 will provide quantitative data, however, the optimal amount of template DNA may need to be optimized empirically. Chicken GAPDH primers are used as an internal control The PCR is run under the following conditions for Alu sequences: polymerase activation at 95&deg;C for 2 min followed by 30 cycles at 95&deg;C for 30 s., 63&deg;C for 30 s., 72&deg;C for 30 s. The reaction conditions for chicken GAPDH are identical to those described for Alu however it may be necessary to extend the PCR to 40 cycles.</li>
<li>Quantitative analysis of metastasis is performed using the &Delta;&Delta;Ct method comparing experimental groups to a control group (Schmittgen and Livak, 2008; Zijlstra et al., 2002). Alternatively a standard curve can be generated using a dilution series of human tumor cells extracted using the Extract-N-Amp kit (Zijlstra et al., 2002). Statistical significance is evaluated using Student T-test or Anova analysis of control and experimental groups.</li> 
</ol>
8. Representative Results:
<img src="/files/ftp_upload/2815/2815fig1.jpg" alt="Figure 1"/> 
Figure 1. The chicken embryo metastasis model. A) A four panel diagram illustrating the appearance of the model in cross section at key steps: 1. After 10 days of incubation. The CAM has reached near-maximum size. The arteries and veins are distinctly visible with the Allantoic vein positioned against the top of the egg. 2. The egg immediately after drilling two holes in the eggshell: one into the airsac and one adjacent to the attachment point for the allantoic vein. 3. The appearance after a mild vacuum has been used to evacuate the airsac and drop the CAM. Tumor cells (blue) are applied to the dropped CAM. 4. After allowing the grafted tumor cell to grow, the tumor along with tissues from the chicken embryo are harvested for analysis. B) A serial depiction of the model: 1. Incubating the eggs., 2. Marking the location of the allantoic vein., 3. Drilling holes in the eggshell., 4. Dropping the CAM., 5. Cutting an opening for tumor cell grafting., 6. Applying the tumor cells., 7. Cooling the eggs on ice prior to collecting tissue., 8. Harvesting the tumor after 7 days of growth.
<img src="/files/ftp_upload/2815/2815fig2.jpg" alt="Figure 2"/> 
Figure 2. Histology of CAM tumors. Tissues taken from tumor bearing chorioallantoic membrane was fixed in formalin, sectioned and processed with a trichrome stain. A) HEp3 tumor after seven days of growth on the CAM. B and C show magnified regions from A showing both tumor and stroma. D) normals CAM.
<img src="/files/ftp_upload/2815/2815fig3.jpg" alt="Figure 3"/> 
Figure 3. Quantitative longitudinal analysis of metastasis. Variants for the head and neck carcinoma HEp3 with high or low metastatic ability (HEp3 M(High) and HEp3 M(Low) respectively) were analyzed for their ability to disseminate using Alu PCR. Ten animals were analyzed at each time point for the duration of 7 days. These samples were quantified against a standard curve and subsequently expressed as #cells/50mg tissue.
<img src="/files/ftp_upload/2815/2815fig4.jpg" alt="Figure 4"/> 
Figure 4. The inhibition of spontaneous metastasis cells in animals treated with the metastasis inhibiting antibody 1A5. Tumors were formed using 5X105 HEp3 cells applied to the CAM of day-10 embryos. Three days after applying the tumor cells half the animals were treated with the anti-CD151 antibody 1A5. The lungs from tumor-bearing animals were harvested two days later and subjected to quantitative real-time alu PCR. Conversely quantitative real-time PCR of chGAPDH was used to confirm the presence of equivalent quantities of host genomic DNA (The &Delta;&Delta;Ct method was used to compare the two groups quantitatively (B).
<img src="/files/ftp_upload/2815/2815fig5.jpg" alt="Figure 5"/> 
Figure 5. Evaluating arrest, growth, and intravasation in the metastatic cascade. Fundamentally, metastasis to a secondary organ is defined by the contributions of three aspects: arrest, rate of growth, and rate of intravasation. These can be quantified using a combination of experimental metastasis (A) and spontaneous metastasis (B). Using experimental metastasis (A) arrest is defined as the number of cell detected 2hr post injection. Survival and growth of arrested cells is determined as the change in cell number 24hr post injection. Growth is defined as the rate of proliferation during days 3-7. Intravasation is determined using spontaneous metastasis (B) where the number intravasated cells is defined as the metastatic burden present on day 7 that exceeds the value predicted by the growth of the metastatic burden present on day 6.
<img src="/files/ftp_upload/2815/2815fig6.jpg" alt="Figure 6"/> 
Figure 6. Visualizing expansion of the metastatic in the CAM. HEp3 cells expressing GFP were injected intravenously on developmental day 12. On day 1, 3, and 5 an egg was sacrificed and a section of CAM was imaged with a fluorescent stereo microscope. The dark green background is created by the eggshell, the vasculature is visible as black lines, and the tumor cells are visible as bright green spots.

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+metastatic+properties+of+a+variety+of+mouse,+rat,+and+human+cells+in+assays+in+nude+mice+and+chick+embryos.">Comparison of metastatic properties of a variety of mouse, rat, and human cells in assays in nude mice and chick embryos.</a> In Vivo. 4, 215-219 (1990).</li><li>Deryugina, E. I., Zijlstra, A., Partridge, J. J., Kupriyanova, T. A., Madsen, M. A., Papagiannakopoulos, T., Quigley, J. 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No conflicts of interest declared.

The chick CAM has been used for decades as a model system to study various aspects of cancer biology and metastatic progression. Principle evaluation of metastatic abilities have been performed in this model by a variety of groups including the analysis of dormancy (Aguirre Ghiso et al., 1999; Ossowski and Reich, 1983), tissue remodeling (Deryugina et al., 2005; Hahn-Dantona et al., 1999), and metastasis (Zijlstra et al., 2008; Zijlstra et al., 2002). It continues to be a very...

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>1X Dulbecco's Modified Eagle Medium (DMEM)</td>
 <td>Invitrogen</td>
 <td>11995073</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Dulbecco's Phosphate-Buffered Saline (D-PBS) (1X), liquid</td>
 <td>Invitrogen</td>
 <td>14190250</td>
 <td>pH 7.4</td>
 </tr>
 <tr>
 	<td>Trypsin, 0.05% (1X) with EDTA 4Na, liquid</td>
 <td>Invitrogen</td>
 <td>25300054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Sportsman hatcher</td>
 <td>Berry Hill</td>
 <td>1550HA</td>
 <td rowspan="2">These incubators are some of the most readily available. However, the video will show different equipment.</td>
 </tr>
 <tr>
 	<td>Sportsman incubator </td>
 <td>Berry Hill</td>
 <td>1502EA</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Dremel rotary tool</td>
 <td>Dremel</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Dremel cutoff wheels no. 36 </td>
 <td>Dremel</td>
 <td>409</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Portable Pipette Aid</td>
 <td>Fisher</td>
 <td>1368115E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Cotton Tipped Applicators</td>
 <td>Fisher</td>
 <td>23-400-001)</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fertilized Eggs</td>
 <td>Various</td>
 <td>&nbsp;</td>
 <td>Many different vendor can be used. The eggs should be approximately 35-55 grams. The hens that lays them should be between 36 and 55 weeks of age.</td>
 </tr>
 <tr>
 	<td>Hemocytometer</td>
 <td>Hausser Scientific, VWR</td>
 <td>15170-090</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Fiber-optic microscope illuminator</td>
 <td>Amscope</td>
 <td>HL250-AY</td>
 <td>150W</td>
 </tr>
 <tr>
 	<td>25 gauge needle</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Sybr Green Extract&#x2013;N-Amp Tissue PCR Kit</td>
 <td>Sigma-Aldrich</td>
 <td>XNATRG</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

quantitative analysis of cancer metastasis using an avian embryo model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Watch this Scientific Journal Video about Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model at JoVE.com

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

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18.9K Views.  Vanderbilt University. Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Video: Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

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 ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

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 ...

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  ...

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 ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...