Name: in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model
Uploaded: 2011-10-03T13:00:00
Description: Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

This study is supported in part by DOD Breast Cancer IDEA Award W81XWH-08-1-0583 and NIH/NCI CA141348-01A1 (DZ) and FAMRI clinical scientist award (DS). Imaging infrastructure is provided by Southwestern Small Animal Imaging Research Program supported in part by U24 CA126608 and Simmons Cancer Center (P30 CA142543) and NIH 1S10RR024757-01.

All animal procedures were approved by the Institutional Animal Care and Use Committee of University of Texas Southwestern Medical Center.
1. In vitro HIF-1&alpha; bioluminescence assay
<ol>
 <li>Materials and Methods:
 <ul>
 <li>Human metastatic breast cancer cell line MDA-MB231 transfected with a novel HIF-1-dependent reporter gene, 5HRE-ODD-luc was generated by Dr. Harada.</li>
 <li>In hypoxic condition, the enhanced expression of oxygen-dependent degradation domain (ODD)-Lucif...

<ol>
	<li>Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+hypoxia-response+enhancers+and+an+oxygen-dependent+proteolytic+motif+enables+real-time+imaging+of+absolute+HIF-1+activity+in+tumor+xenografts.">The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.</a> Biochem Biophys Res Commun. 360, 791-796 (2007).</li><li>Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+HIF-1+imaging+for+determining+optimal+timing+of+combining+bevacizumab+and+radiotherapy.">Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy.</a> Int J Radiat Oncol Biol Phys. 75, 463-467 (2009).</li><li>Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, e8051-e8051 (2009).</li><li>Contero, A., Richer, E., Gondim, A., Mason, R. 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Breast cancer brain metastasis occurs in 30% of breast cancer patients at stage IV. It is associated with high morbidity and mortality and has a median survival of 13 months6. There is a need to have appropriate animal models to mimic this clinically devastating disease in order to facilitate our understanding of its intracranial initiation and progression as well as pathophysiological profiles. Here, we have developed an orthotopic breast cancer brain metastasis model by injecting human breast cancer cells, d...

<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>D-luciferin</td>
<td>Gold Biotechnology </td>
<td>L-123</td>
<td>120 mg/kg in PBS in a total volume of 80 &mu;l for in vivo study</td>
</tr>
<tr>
<td>Isoflurane</td>
<td>Baxter International Inc.</td>
<td>1001936060</td>
<td></td>
</tr>
<tr>
<td>Matrigel</td>
<td>BD Biosciences</td>
<td>354234</td>
<td></td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>Hamilton Company</td>
<td>1701</td>
<td></td>
</tr>
<tr>
<td>32G Hamilton needle</td>
<td>Hamilton Company</td>
<td>7803-04</td>
<td></td>
</tr>
<tr>
<td>Hypoxia chamber</td>
<td>Billups-Rothenberg, Inc.</td>
<td>MIC-101 </td>
<td></td>
</tr>
<tr>
<td>Bioluminescence imaging system</td>
<td>Caliper Life Sciences</td>
<td>IVIS Spectrum system</td>
<td></td>
</tr>
<tr>
<td>G418</td>
<td>Fisher scientific</td>
<td>SV3006901</td>
<td></td>
</tr>
</tbody>
</table>

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.

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

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.

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 ...

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