Name: mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression
Uploaded: 2012-02-17T12:00:00
Description: Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

This work was supported, in part, by National Institutes of Health R01 CA131990 (BFS and RRM). Live-cell imaging was performed in the Microscopy, Imaging and Cytometry Resources Core supported, in part, by NIH Center grant P30 CA22453 to the Karmanos Cancer Institute, Wayne State University and by the Perinatology Research Branch of the National Institutes of Child Health and Development, Wayne State University.

1. Prepare DQ-substrates
<ol>
	<li>Allow lyophilized DQ-substrates to warm to room temperature before opening vials, prepare stock solution of 1 mg/ml of DQ-substrate in deionized water, divide into 50 &mu;l aliquots and store at 4 &deg;C.</li>
</ol>
It may be necessary to agitate DQ-substrate in an ultrasonic water bath for ~5 min and heat to 50 &deg;C to facilitate dispersion.
<ol start="2">
	<li>Thaw rBM on ice overnight at 4 &deg;C; rBM should be handled on ice at all time...

<ol>
	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

As demonstrated, the MAME cocultures can be used for live-cell imaging in real-time of interactions among the various cellular constituents that comprise a breast tumor and its microenvironment. In ongoing studies in our laboratory, we have used MAME cocultures to identify proteolytic pathways associated with transition from DCIS to invasive ductal carcinoma, as well as the interactions between proteolytic pathways and other pathways involved in this transition such as chemokine/cytokine/growth factor pathways. We furthe...

<table border="1">
<tbody>
 <tr>
 <td>Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Reconstituted basement membrane (Cultrex)</td>
 <td>Trevigen</td>
 <td>3445-005-01</td>
 <td>Comparable to Matrigel (BD Biosciences)</td>
 </tr>
 <tr>
 <td>Collagen I</td>
 <td>Cohesion Laboratories</td>
 <td>5005-B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DQ-substrates 
 (collagen I, IV)</td>
 <td>Invitrogen</td>
 <td>DQ-col I-D12060 
 DQ-col&nbsp;IV&nbsp;D12052</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>22-mm plastic coverslips</td>
 <td>Fisher Scientific Co. </td>
 <td>12-547</td>
 <td>Cut in half, leave in 70% ethanol for 10 min and air-dry before use.</td>
 </tr>
 <tr>
 <td>Cell culture medium</td>
 <td>Lonza</td>
 <td>MEBM-PRF&nbsp;CC-3153 
 MEGM CC-4136</td>
 <td>Phenol red&ndash;free</td>
 </tr>
 <tr>
 <td>ibiTreat &mu;-Dish</td>
 <td>Ibidi</td>
 <td>80136</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Volocity software</td>
 <td>Perkin-Elmer</td>
 <td>Version 5.5</td>
 <td>Other brands of imaging software can be used to generate 3D reconstructions and movies.</td>
 </tr>
 </tbody>
</table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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