The authors acknowledge the following funding sources: P01CA040035 (FE/JAS) and VA Career Development Award (JAS).

1. Cell Maintenance
<ol>
 <li>Many bone metastatic subclones of MDA-MB-231 exist that have variable propensity for bone colonization and growth. For our experiments we use a clone derived from the GFP-tagged MDA-MB-231 developed at UTHSCA 2. This particular sub-clone will form visible osteolytic bone lesions at approximately 3 weeks post inoculation, with sacrifice at 4 weeks.</li>
 <li>Maintain cells in DMEM containing 10% FBS and 0.1 mg/ml penicillin-streptomycin at 37 &deg;C in 5-8% CO27.</li>
 <li>Passage cells at 80-90% confluence and re-plate at a 1:10 dilution (this varies depending on cell type and the size plate. For MDA-MB-231 cells in a T-75 this is approximately 6x105), which is approximately every 72 hr.</li>
 <li>MDA-MB-231 cells maintain a generally fibroblastic appearance with numerous pseudopodia. Any rounding is indicative of overcrowding or other undesirable stress, and should be avoided since this may affect metastatic potential in vivo.</li>
 <li>Ideally, cells should be maintained in culture only briefly before inoculation (we recommend less than 1 wk or until you have enough cells for the experiment), and a new vial should be thawed if rounding or changes in cell growth rate occur.</li>
</ol>
2. Cell Preparation
<ol>
 <li>Trypsinize cells at 80-90% confluence with 0.15% Trypsin/EDTA (dilute 0.5% Gibco Trypsin EDTA 1:2 in PBS). This concentration allows for fast detachment (&lt;2 min) and reduces clumping of the cells.</li>
 <li>Immediately remove cells from plate with 10 ml ice-cold DMEM containing 10% FBS, pipette into 15 ml conical tube and centrifuge at 200 x g for 5 min.</li>
 <li>Resuspend pellet in a 50 ml conical tube in 25 ml ice-cold PBS (without Ca and Mg) and count.</li>
 <li>Centrifuge to re-pellet, and resuspend at 106 cells per ml (intracardiac) or at 250,000 cells/10 &mu;l (intratibial) in ice-cold PBS. The temperature of PBS is important to prevent clumping of cells and subsequent embolism after injection. The cell suspension should remain on ice while performing injections and will be viable and remain un-clumped for 30 min. While the exact number of plates required will vary from investigator to investigator, we generally use 1 T-75 for between 8-10 mice.</li>
</ol>
3. Intracardiac Inoculation
<ol>
 <li>4-6 week old female (Foxn nu-/-: Harlan) mice are used for these experiments, though other immune-compromised mice (RAG1-/-, RAG2-/-, SCID, XID, and etc.) can also be used with human cancer cell experiments.</li>
 <li>The nude mice should be fed Teklad 2920x diet 5-10 days before injection to decrease mortality from the injection procedure, and reduce background fluorescence in imaging procedures (since the food is alfalfa free).</li>
 <li>If using mice other than nu/nu, remove hair by shaving or using Nair (in our experience Nair works better) on ventral abdominal area before beginning procedure.</li>
 <li>Anesthetize mouse in an isolation chamber using an isoflurane vaporizer (2.5% Iso: 2-3 L/min O2) or other preferred anesthesia (in our experience isoflurane is the best tolerated for intracardiac inoculations).</li>
 <li>Move one mouse at a time to the nose cone of the anesthesia machine inside a stainless steel ventilated hood.</li>
 <li>Position each mouse on its back with chest facing up.</li>
 <li>Wash the chest with a 10% povidone/iodine swab/solution followed by 70% ethanol, repeating 2 times.</li>
 <li>Mark mouse's chest for injection (using a sterile marker or marking slightly to the side to keep the injection site sterile). We use a mark location midway between the sternal notch and top of xyphoid process, and slightly left (anatomical) of the sternum. Draw a small bubble of air into syringe to create space between the plunger and meniscus (important for seeing cardiac pulse) of a 300 &mu;l 28 g &frac12; insulin syringe, and draw up 100 &mu;l of cells.</li>
 <li>Keep needle upright. While holding skin of mouse taut with other hand, insert needle.</li>
 <li>Successful insertion into left cardiac ventricle should result in a distinct bright red pulse of blood in the syringe. At this depth, carefully depress plunger of syringe (100 &mu;l volume), without significant movement of the needle to avoid puncturing the heart or spilling cells into the chest cavity.</li>
 <li>After cells are injected, pull up slightly on plunger to create a small amount of negative pressure to reduce dripping of cells into the chest cavity.</li>
 <li>Pull needle directly out of chest while being sure to avoid tilting the needle during removal, which can tear the lining of the heart and cause bleeding.</li>
 <li>Apply gentle pressure to the chest over the injection site to reduce bleeding.</li>
 <li>Remove mouse from the nose cone and continue to apply light pressure for about 1 min.</li>
 <li>Move mouse to heating pad until fully conscious.</li>
 <li>If mouse begins to spin or twitch after recovery, the likely cause is an embolism. Inject mouse intraperitonally with 80-120 mg/kg of ketamine to increase blood flow and cardiac output while decreasing vascular resistance. This should help the mouse pass the clot. After this occurs, it is best to change cell preparations, as it indicates that the cells are clumping. We change cell preparations after approximately 8 mice, or 30 min.</li>
</ol>
4. Intratibial
<ol>
 <li>Inject mouse with Buprenex (0.1 mg/kg) or similar approved analgesic immediately before procedure.</li>
 <li>Anesthetize mouse using isoflurane, then move to nose cone and maintain anesthesia.</li>
 <li>Clean both legs with 10% povidone/iodine swab/solution, followed by ethanol, repeating 2 times. Depilate legs (with Nair or similar product) prior to cleaning if fur present.</li>
 <li>Gently grasp lateral malleolus, medial malleolus, and lower half of tibia with forefinger and thumb, then bend leg (combination of flexion and lateral rotation, such that the knee is visible and accessible.</li>
 <li>Wet the skin with 70% EtOH to increase visibility of underlying patellar ligament, which should be visible as a distinct, thick, white line. While firmly grasping ankle/leg of mouse insert 28g &frac12; needle under patella, through the middle of patellar ligament, and into the anterior intercondylar area in top of tibia.</li>
 <li>When inserting needle into tibia, guide carefully through growth plate using steady, firm pressure with slight drilling action.</li>
 <li>Upon penetration of tibial growth plate, the needle will encounter markedly less resistance.</li>
 <li>Use a gentle, lateral movement of needle to ensure needle is in tibia and through the growth plate. Movement will be limited if needle is in proper place within tibia.</li>
 <li>Slowly depress plunger to inject 10 &mu;l of cell solution. Little to no resistance should be felt at this point.</li>
 <li>Slowly extract needle.</li>
 <li>Following the same procedure, proceed to next leg to inject 10 &mu;l of PBS.</li>
 <li>Remove mouse from anesthesia and keep on heating pad until recovered.</li>
 <li>Monitor the mice over the next 24 hr and inject with additional Buprenex every 12 hr if animals continue to show signs of distress.</li>
</ol>
5. Imaging Time Course
<ol>
 <li>Luciferase Imaging: Where applicable, luciferase imaging should be utilized beginning at 7 days post-tumor cell inoculation (though in most bone metastasis models, detectable closer to day 14). Mouse is injected intraperitoneally with 150 mg/kg luciferin, anesthetized, and imaged 8 min after injection using IVIS equipment 8.</li>
 <li>GFP imaging: Typically GFP-expressing tumors begin to be detectable around day 10-14 for intratibial injections and 16-21 for intracardiac experiments using Maestro imaging equipment (CRi) 9. Briefly, mice are anesthetized with isoflurane (3%) and maintained under anesthesia through a nose cone in the Maestro imaging chamber. For our eGFP-expressing cells we use the blue filter set (498 excitation/515 emission for eGFP) at 500 ms exposure in 10 nm steps.</li>
 <li>Faxitron (X-ray): X-rays are taken weekly starting between day 7 to 14. Lesions will be visible in the intracardiac model at day 21 and a week earlier in the intratibial injections3. Our radiographic data for ex vivo and in vivo was acquired using a Faxitron LX-60 at 35 kV for 8 seconds of exposure.</li>
 <li>Other Imaging: Imaging modalities will be entirely based upon the specific research. In addition to the above methods, live animal imaging based on 3D micro-computed tomography (&mu;CT), micro-positron emission tomography (&mu;PET), and other modalities 10,11 can provide valuable information.</li>
</ol>
6. Mouse Sacrifice
<ol>
 <li>Each model has a slightly different time course. With MDA-MB-231 cells mice usually become cachectic or paraplegic between 21 and 35 days and usually around day 28 (IT injected mice between 21-28 days).</li>
 <li>Once several mice develop lesions that begin to break through the cortical bone, become paraplegic, or lose between 10-20% of body weight, sacrifice all mice in study using ketamine/xylazine overdose with cervical dislocation (or other IACUC approved method).</li>
 <li>Remove bones needed for further study and store in buffered 4% formalin.</li>
 <li>Mice with chest tumors are a sign of a missed injection and should be excluded from the study.</li>
</ol>
7. Ex Vivo Analysis
<ol>
 <li>&mu;CT: scanning in 70% EtOH allows for structural and histological parameters to be measured on the same bone specimen.</li>
 <li>Contour and analyze according to individual experimental design and available equipment10. Our data was obtained with a Scanco &mu;CT 40 using 12 &mu;M resolution.</li>
 <li>Histomorphometry: Process specimens using preferred histological methods and stain with Hematoxylin/Eosin7</li>
</ol>
8. Representative Results
Following intracardiac or intratibial inoculation of tumor cells (Figure 1) mice are monitored weekly by radiography and fluorescence (or luminescence). Lesions normally become apparent by x-ray between week 2 and 3, while they may be visible by fluorescence and luminescence as early as week 2, depending on the model (Figure 2). Lesions are visible as dark holes in the bone, and become more pronounced with time (Figure 3). Lesions were quantified using Metamorph analysis of radiographs and fluorescence was quantified using Maestro (CRi) imaging acquisition and analysis software. As lesions begin to grow, mice frequently become cachectic. Sacrifice is required when mice have lost between 10-20% of their initial body weight. The condition of the mice can change rapidly and must be monitored multiple times during the day. Between 3-4 weeks mice began to drag one or both hind limbs and the entire study was sacrificed. Images were taken prior to sacrifice. After anesthesia overdose, blood was collected to measure markers of bone turnover or other factors. After cervical dislocation, the skin was stripped from the mouse and bones excised and cleaned for ex vivo imaging (Figure 3) which will allow visualization of tumor foci at least 7 days earlier than in vivo imaging techniques. Once imaging was completed bones were placed in labeled cassettes and stored in formalin for fixation and further histological processing or &mu;CT analysis. Histomorphometry was performed on the samples to indicate the tumor burden and BV/TV after &mu;CT was performed on a tibia to quantify the amount of bone destruction (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/4260/4260fig1.jpg" /> 
 Figure 1. Proper placement of injection. Ventral radiographical view (A) of adult mouse thorax showing proper placement of needle in the 4th intercostal space and into the left ventricle for intracardiac inoculation. Anatomical landmarks are shown with horizontal dashed lines on the sternum (outlined in white). On the mouse's skin the sternal notch and xyphoid process serve as landmarks, and the needle is inserted slightly left of the sternum. Ventral radiographical view (B)of adult mouse leg showing proper placement of needle in the proximal tibia, aligned between the condyles. From a lateral perspective the tip of needle is placed behind the tibial tuberosity and deep to the epiphyseal plate. Ideal location for injection is shown by white circle.
<img alt="Figure 2" src="/files/ftp_upload/4260/4260fig2.jpg" /> 
 Figure 2. Time course of bone metastasis models. Progression of cancer-induced bone disease shown with x-rays and GFP+ fluorescent images of the tumors in vivo from intratibial injections (A) and intracardiac injections (B).
<img alt="Figure 3" src="/files/ftp_upload/4260/4260fig3.jpg" /> 
 Figure 3. Time course of intracardiac MDA-MB-231 model. Top Row: Ex vivo image showing GFP fluorescence at day 0, 14, 21, and 28 after intracardiac injection of MDA-MB-231. Note that tumor foci at day 14 are only visible in ex vivo after soft tissue removal. Bottom row: Ex vivo radiographic images of the same femora illustrating osteolytic bone lesions. Note that bone destruction is not usually visible until 3 weeks after cardiac injection.
<img alt="Figure 4" src="/files/ftp_upload/4260/4260fig4.jpg" /> 
 Figure 4. Quantification of bone metastatic progression. GFP+ tumor as visualized by Maestro imaging ex vivo of tibiae of a non-tumor bearing mouse (A) and a mouse 4 weeks after intracardiac injection of a GFP expressing MDA-MB-231 subclone (B). Tumor foci correlate with areas of bone destruction as shown by Faxitron (D) compared a bone without tumor (C). Bone structural and mineral data from microCT measurements is rendered as 3d images of a healthy bone (E) and a tumor bearing bone (F) where areas of bone destruction are shown as void area. Further bone and tumor detail is seen in corresponding H&amp;E stained histological sections (G &amp; H).

<ol>
	<li>Sterling, J. A., Edwards, J. R., Martin, T. J., Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+biology+of+bone+metastasis:+how+the+skeleton+affects+tumor+behavior.">Advances in the biology of bone metastasis: how the skeleton affects tumor behavior.</a> Bone. 48, 6-15 (2011).</li><li>Yoneda, T., Sasaki, A., Mundy, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteolytic+bone+metastasis+in+breast+cancer.">Osteolytic bone metastasis in breast cancer.</a> Breast Cancer Res. Treat. 32, 73-84 (1994).</li><li>Johnson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+promotion+of+Gli2-induced+expression+of+parathyroid+hormone-related+protein,+an+important+osteolytic+factor+in+bone+metastasis,+is+independent+of+canonical+Hedgehog+signaling.">TGF-beta promotion of Gli2-induced expression of parathyroid hormone-related protein, an important osteolytic factor in bone metastasis, is independent of canonical Hedgehog signaling.</a> Cancer Res. 71, 822-831 (2011).</li><li>Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+TGF-beta+Responsiveness+in+Prostate+Stromal+Cells+Alters+Chemokine+Levels+and+Facilitates+the+Development+of+Mixed+Osteoblastic/Osteolytic+Bone+Lesions.">Loss of TGF-beta Responsiveness in Prostate Stromal Cells Alters Chemokine Levels and Facilitates the Development of Mixed Osteoblastic/Osteolytic Bone Lesions.</a> Mol. Cancer Res. , (2012).</li><li>Yoneda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actions+of+bisphosphonate+on+bone+metastasis+in+animal+models+of+breast+carcinoma.">Actions of bisphosphonate on bone metastasis in animal models of breast carcinoma.</a> Cancer. 88, 2979-2988 (2000).</li><li>Rose, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoactivin+promotes+breast+cancer+metastasis+to+bone.">Osteoactivin promotes breast cancer metastasis to bone.</a> Mol. Cancer Res. 5, 1001-1014 (2007).</li><li>Sterling, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hedgehog+signaling+molecule+Gli2+induces+parathyroid+hormone-related+peptide+expression+and+osteolysis+in+metastatic+human+breast+cancer+cells.">The hedgehog signaling molecule Gli2 induces parathyroid hormone-related peptide expression and osteolysis in metastatic human breast cancer cells.</a> Cancer Res. 66, 7548-7553 (2006).</li><li>Lu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VCAM-1+promotes+osteolytic+expansion+of+indolent+bone+micrometastasis+of+breast+cancer+by+engaging+alpha4beta1-positive+osteoclast+progenitors.">VCAM-1 promotes osteolytic expansion of indolent bone micrometastasis of breast cancer by engaging alpha4beta1-positive osteoclast progenitors.</a> Cancer Cell. 20, 701-714 (2011).</li><li>Oyajobi, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+myeloma+in+skeleton+of+mice+by+whole-body+optical+fluorescence+imaging.">Detection of myeloma in skeleton of mice by whole-body optical fluorescence imaging.</a> Mol. Cancer Ther. 6, 1701-1708 (2007).</li><li>Johnson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+live+animal+micro-CT+allows+for+quantitative+analysis+of+tumor-induced+bone+destruction.">Longitudinal live animal micro-CT allows for quantitative analysis of tumor-induced bone destruction.</a> Bone. 48, 141-151 (2011).</li><li>Sterling, J., Johnson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaging Techniques for the Detection and Examination of Breast Cancer Metastasis to Bone. 46, (2011).</li><li>Steinbauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-transfected+tumor+cells+are+useful+in+examining+early+metastasis+in+vivo,+but+immune+reaction+precludes+long-term+tumor+development+studies+in+immunocompetent+mice.">GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice.</a> Clin. 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No conflicts of interest declared.

Studies of tumor-induced bone disease rely on several animal models of which the two most commonly used are intracardiac and intratibial inoculations. In many cases intracardiac is the best option for studying metastasis to bone; however, it does not represent the full metastatic process, and therefore mammary fat pad or other orthotopic injections should ideally be used toward that end. Tumor cells do not metastasize readily from the primary site in most studies using human cells, and if so, they primarily metastasize t...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>MDA-MB-231 human breast cancer cells</td>
 <td>Originally ATCC-Internally cloned and selected</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>GIBCO</td>
 <td>11885</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Mediatech Inc</td>
 <td>21-040-CV</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Atlas Biological</td>
 <td>F-0050a</td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>GIBCO</td>
 <td>15400</td>
 </tr>
 <tr>
 <td>Faxitron</td>
 <td>Faxitron</td>
 <td></td>
 </tr>
 <tr>
 <td>Maestro</td>
 <td>CRi</td>
 <td></td>
 </tr>
 <tr>
 <td>&mu;CT scanner</td>
 <td>Scanco</td>
 <td></td>
 </tr>
 <tr>
 <td>Athymic Nude Mice</td>
 <td>Harlan</td>
 <td>Fox nu -/-</td>
 </tr>
 <tr>
 <td>Insulin Syringes (300 &mu;l, 28.5 g)</td>
 <td>Becton Dickinson</td>
 <td>309300</td>
 </tr>
 <tr>
 <td>IVIS</td>
 <td>Caliper</td>
 <td></td>
 </tr>
 <tr>
 <td>Irradiated Rodent Diet</td>
 <td>Teklad</td>
 <td>2920x</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Models of Bone Metastasis at JoVE.com

Models of Bone Metastasis

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 ...

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41.9K Views.  Vanderbilt University. 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.

Video: Models of Bone Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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 ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

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  ...

Adjustable Stiffness, External Fixator for the Rat Femur Osteotomy and Segmental Bone Defect Models

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there has been great clinical interest in improving bone healing by altering the mechanical environment through the fixation stability around the lesion. One constraint of preclinical animal research in this area is the lack of experimental control over the local mechanical environment within a large segmental defect as well as osteotomies as they heal. In this paper we report on the design and use of an external fixator to study the healing of large segmental bone defects or osteotomies. This device not only allows for controlled axial stiffness on the bone lesion as it heals, but it also enables the change of stiffness during the healing process in vivo. The conducted experiments have shown that the fixators were able to maintain a 5 mm femoral defect gap in rats in vivo during unrestricted cage activity for at least 8 weeks. Likewise, we observed no distortion or infections, including pin infections during the entire healing period. These results demonstrate that our newly developed external fixator was able to achieve reproducible and standardized stabilization, and the alteration of the mechanical environment of in vivo rat large bone defects and various size osteotomies. This confirms that the external fixation device is well suited for preclinical research investigations using a rat model in the field of bone regeneration and repair.

One constraint of preclinical research in the field of bone repair is the lack of experimental control over the local mechanical environment within a healing bone lesion. We report the design and use of an external fixator for bone repair with the ability to change fixator stiffness in vivo.

The mechanical environment around the healing of broken bone is very important as it determines the way the fracture will heal. Over the past decade there ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

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 ...

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.

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 ...