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</...

<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. Exp. Metastasis. 20, 135-141 (2003).</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=Dual+effects+of+macrophage+inflammatory+protein-1alpha+on+osteolysis+and+tumor+burden+in+the+murine+5TGM1+model+of+myeloma+bone+disease.">Dual effects of macrophage inflammatory protein-1alpha on osteolysis and tumor burden in the murine 5TGM1 model of myeloma bone disease.</a> Blood. 102, 311-319 (2003).</li></ol>

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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